Enabling water-based high-density nanoparticles assembly by using silk fibroin as an adsorbate

Domain: physics
Published: 2026-02-10
Editorial outcome: minor_revision

Abstract
Water-based fabrication holds promise for innovations in life sciences, electronics, and materials science at the biotic-abiotic interface by integrating living systems with high technologies. However, using water to create bio-nano interfaces is challenging, as it often requires surface pre-treatment and thermal processing, which are both harmful to living systems. Here, we propose silk fibroin (SF) as a natural adsorbate that enables the fabrication of high-density nanoparticle (NP) layers relying solely on water-based processing. We show that SF spontaneously adsorbs onto various NPs, enhancing intermolecular interactions and facilitating the wetting of otherwise hydrophobic substrates, resulting in densely packed NP layers. Several SF-adsorbed NP electronics are demonstrated with performance comparable to their conventional counterparts. This work offers significant utility by establishing a flexible and biocompatible approach for the fabrication of seamless, precisely controlled bio-nano interfaces.

Introduction
The biotic-abiotic interface and the integration of living systems with conventional nanotechnologies are critical for advancing next-generation devices that seamlessly interface biological systems with engineered materials. This enables real-time sensing, adaptive functionality, and sustainable operation within living environments. This convergence is foundational for innovations in bioelectronics, data storage, personalized medicine, and environmentally responsive technologies1–4. However, beyond addressing fundamental mismatches in mechanical properties and communication modalities between these domains5–7, a critical challenge lies in preserving the intrinsic biological functions of labile and vulnerable biogenic substances when establishing such interfaces8–10. Achieving this requires biocompatible fabrication approaches that not only maintain biochemical functionality but also meet the high standards of resolution and precision inherent in cutting-edge nanotechnology, ensuring mutual compatibility and functionality in both domains.

Water-based nanofabrication is a biocompatible approach for high-resolution processing that can bridge the challenging gap between biological systems and advanced technologies. However, water’s intrinsic physical properties, such as high surface tension and low volatility, pose significant challenges for surface wetting, which is necessary to form reliable interfaces and fabricate high-quality devices. Conventionally, these issues are typically addressed through surface pre-treatments (e.g., plasma, UV/Ozone, and acid/base treatments) to enhance interfacial wetting, followed by high-temperature annealing to remove organic residues and increase the crystallinity, thereby creating high-density and defect-free films11,12. Unfortunately, both approaches are generally incompatible with most biological substances.

Extensive foundational studies have established the physico-chemical principles governing protein adsorption (e.g., silk fibroin (SF)) under aqueous conditions, including its effects on wettability, secondary-structure transitions, and biological or material coating13–18. While these works suggest that protein adsorption may offer a natural route for forming biocompatible interfaces, they focus primarily on fundamental interfacial behavior. Building upon this knowledge, recent efforts have begun translating such phenomena into aqueous nanoprocessing strategies19,20. However, these approaches still rely on high-temperature annealing or solvent activation. A fully water-based route capable of producing high-density, electronically functional nanoparticles (NPs) layers without post-thermal processing remains unaddressed.

NPs can exhibit distinct functionalities when their size and composition are precisely controlled21. They can be directly dispersed in water through surface functionalization22, and their biocompatibility can be further enhanced by appropriate surface passivation23. Therefore, improving the interfacial wettability of water-dispersible functional NPs to achieve uniform surface coverage while simultaneously forming high-density NPs layers without compromising their intrinsic properties can be a promising strategy to overcome existing technological challenges in water-based fabrication processes aimed at developing high-performance bio-nano interfaces.

We show here that SF can function as a natural amphiphilic adsorbate to enable water-based fabrication of high-density NP layers while preserving the original functionality of the NPs. When added to aqueous NP dispersions, SF spontaneously forms a nanoscopic adsorption layer around various NPs through multiple interaction routes (e.g., van der Waals, electrostatic, and hydrogen bonding interactions)24,25. Analysis of adsorption behavior of SF and its role in enhancing intermolecular interactions between NPs during the coating phase points that the conformational rearrangement and network entanglement of adsorbed fibroin chains upon physical contact are key mechanisms enabling the self-assembly26–28. These augmented interactions allow aqueous NP dispersions containing a small, precisely controlled amount of SF (~0.2 w/v%) to effectively wet hydrophobic substrates, such as polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE), resulting in the densely packed NP layers. Additionally, control over the polymorphism of the SF adsorption layer offers further benefits in the sequential stacking of water-based NP dispersions. Through this physico-chemical approach, high-density NP-based electronics can be formed on hard-to-wet surfaces via simple water processing without outcomes that retain the original functionalities of the NPs. These NP-based electronics exhibit electrical performances comparable to those of conventional inorganic solution-processed devices, with no observable degradation due to the presence of the adsorbed SF dielectric.

Results
Concentration-dependent adsorption of SF onto NPs
SF adsorbs spontaneously onto various NPs in a concentration-programmable manner, forming a controllable nanometric adsorption shell. This adsorption reshapes key NP surface characteristics, establishing the physico-chemical basis for the improved wetting and enhanced NP assembly (Fig. 1). This behavior arises from the amphiphilic structure of the SF chain, which allows it to function as a versatile natural surfactant19,29. As illustrated in Fig. 1a, such adsorptions can occur through various interaction routes24,25. This study explores the evolution of SF adsorption onto water-dispersed NPs with varying SF concentration (Fig. 1b). This study focuses on the adsorption-mediated NP-assembly behavior of SF within the optimal concentration regime required for electronic functionality, rather than benchmarking against conventional surfactants whose effects on wettability and rheology have been addressed previously19.

The adsorption behavior of SF onto monodisperse, spherical polystyrene (PS) NPs (⌀~100 nm) is investigated via electron microscopy. Figure 1c illustrates the formation, growth, and saturation of SF adsorption layers on NP surfaces as the SF concentration is gradually increased (red arrows in Fig. 1d). Even at a low concentration of 0.02 w/v%, a thin SF adsorption layer (~3 nm) forms around the NPs. These observed features were neither visible in the pristine transmission electron microscopy (TEM) grids nor in the original NPs (see Supplementary Fig. 1). Additionally, capillary bridges (blue arrows in Fig. 1d) are observed between SF-adsorbed NPs as a result of intimate contact in the dispersed phase, followed by separation during the drying phase. As the amount of SF is increased in the dispersion, the adsorption layer thickens, reaching saturation. In dispersions with 0.2 w/v% SF, a thicker SF adsorption layer (~10 nm) with wider necks in the capillary bridges can be observed. With higher SF concentrations (i.e., 2.0 w/v%), the NP assembly appears fully encapsulated within the SF matrix. The adsorption behavior occurred regardless of the material composition, surface functionalization, shape, or size of the NPs (see Supplementary Figs. 2, 3). A high magnification scanning electron microscopy (SEM) image and corresponding energy dispersive X-ray spectroscopy (EDS) elemental mapping of nitrogen (Nkα) confirmed that the thin membrane covering NPs and interparticle bridges, which are observed in TEM images, resulted from SF adsorption (see Supplementary Fig. 4).

Please note that, unless otherwise specified, 0.02, 0.2, and 2.0 w/v% SF concentrations are denoted as SF002, SF020, and SF200, respectively, throughout the manuscript.

Chemical surface analyses also confirm that the thin membranes observed over the NP surfaces are SF adsorption layers. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of SF-adsorbed NPs reveals a distinct IR spectrum, showing characteristic peaks of both pure SF films and pure PS NPs (see Supplementary Fig. 5). Specifically, IR absorption mapping at 1650 cm−1, corresponding to the Amide I band (C=O stretching), visualizes the spatial distribution of adsorbed SF on NP surfaces (Fig. 1e). At lower SF concentration (i.e., SF002), weak IR absorption occurs at particle boundaries, indicating thin and sparsely distributed adsorption. In contrast, a substantial IR absorption is observed for NPs with a thicker and more uniform SF adsorption layer. For larger-scale particles (~20 µm), SEM-Raman correlative analysis is performed, clearly identifying the SF presence and providing additional conformational information about the adsorbed SF within these larger capillary bridges (see Supplementary Fig. 6).

The physical effect of SF adsorption on various NPs is confirmed via dynamic light scattering (DLS) analysis (Fig. 1f for PS NPs and Supplementary Fig. 7 for other NPs). As the SF content in aqueous PS NP dispersion (⌀~100 nm) increases, the hydrodynamic radius rises and plateaus by ~0.2 w/v%, and beyond this point, no significant differences are observed (p-value~0.89).

The surface charge of the NPs changes in correspondence with the amount of SF in the dispersion. While the concentration at which hydrodynamic size or zeta potential saturation begins varies between different NP types, they all exhibited increases in size and changes in zeta potential with SF adsorption. As SF concentration increases, the zeta potential of the NPs approaches that of pure SF solution (~−3.17 mV). Additionally, Supplementary Fig. 8 demonstrates that, at saturated SF adsorption level (i.e., SF200), the hydrodynamic radius distribution shifts upward relative to the original NPs.

The SF adsorption-induced change in NP surface energy (Fig. 1g, Supplementary Fig. 9) is confirmed by AFM force spectroscopy. The adhesion force between a diamond-like carbon colloidal probe and SF-adsorbed NPs exhibits a clear non-monotonic dependence on SF concentration: adhesion increases from the pristine (~14.4 nN) and SF002 conditions (~22.3 nN) to a pronounced maximum at SF020 (~32.3 nN) and then decreases again at SF200 (~21.8 nN). This result identifies that there is an optimal adsorption window maximizing the particle-to-particle interaction.

Collectively, these AFM results, together with electron microscopy, IR spectroscopy, and DLS, confirm both the concentration-dependent SF adsorption and its controllable mechanical influence on NPs (i.e., hydrodynamic radii, surface charge, and surface energy). Such tunable surface modification is expected to strongly influence intermolecular interactions among NPs during water-based assembly.

Enhanced intermolecular interactions by SF adsorbate
Significantly, SF adsorption can amplify both interparticle and interfacial adhesion through polymer-mediated interactions (Fig. 2). NPs in solution undergo Brownian motions that bring them into frequent diffusive contact with other particles, SF chains, and interfacial surfaces30,31. However, to quantitatively analyze the adhesion dynamics at the nanoscale interface, a controlled and static approach is required, in which a fixed NP interacts with a hydrophobic solid surface under aqueous conditions at varying SF concentrations (Fig. 2a). Adhesion properties such as adhesion forces and energy dissipation are quantified by analyzing force-distance (FD) curves (Fig. 2b)32. Physical modeling of the approach and retract curves provides additional insights into the local elasticity changes in the interface region (i.e., 0–35 nm from the interface) and the molecular-level mechanics within the SF adsorption layer (see Supplementary Note 1, Supplementary Fig. 10).

Figures 2c, d illustrate the SF-adsorption-dependent adhesion evolution across a 10 μm2 area (N=49 points) with increasing SF concentrations. SF adsorption leads to an initial rise in both adhesion force and energy dissipation, reaching 3.5 nN and 0.32 fJ at SF020, respectively, up from 1.7 nN and 0.12 fJ. However, at higher silk concentrations (i.e., SF200), both adhesion properties are compromised. The measured FD curves for each SF level are provided in Supplementary Fig. 11. In addition, contact time significantly affects the adhesion properties between two solids, as shown in Supplementary Fig. 12. Rapid increase in adhesion is observed when the contact time increases from 0.02 s to 10 s. Beyond 10 s, the adhesion properties begin to level off, indicating that the molecular dynamics within SF adsorption are approaching a steady state.

Concentration- and time-dependent adhesion behaviors can be attributed to the presence of the SF adsorption layer and its polymeric network evolution during physical contact between the NP and the solid surface (see Supplementary Note 2). Under low SF adsorption conditions (i.e., <0.002 w/v%), low adhesion and simple FD curves are observed (Supplementary Fig. 11a). When the SF adsorption is optimal and sufficient time for molecular dynamics within the adsorption layer is allowed, conformational rearrangements and network entanglements within the SF chains become more prominent, contributing effectively to stronger particle interactions (Supplementary Fig. 12). This phenomenon is reflected in increasing persistence and contour lengths in the SF adsorption layer as well as emerging bond rupture peaks (Supplementary Fig. 10d–f), a characteristic commonly observed in adhesion by polymers26–28,32,33. However, when the adsorption layer becomes too thick and stiffer, its reduced ability to conform to surface asperities and the extra viscoelastic energy dissipated in the bulk can diminish the real contact area and weaken interfacial bonding, thereby potentially lowering the measured adhesion strength, as described in the supplementary text and observed in the previous study34. These results highlight the need to precisely control SF concentration to induce the optimal level of adsorption, which can directly enhance the particle assembly during the aqueous coating procedure.

Wetting improvement and high-density NP coatings enabled by optimal SF adsorption
Significantly, the ability to control SF adsorption and fibroin-mediated intermolecular interaction improve the wetting of aqueous NP dispersions and enable high-density, uniform coating of NPs even on low-energy substrates (Fig. 3). Spin-coating of aqueous NP dispersions onto hydrophobic substrates like PDMS or PTFE reveals significant improvement in coating coverage depending on the SF adsorption level (Fig. 3a). Supplementary Figs. 13–15 show that less than SF002 adsorption is insufficient for optimal coating results. In contrast, at 0.02–1.0 w/v% of SF, surface coverage and packing density of NP assembly are gradually improved with the adsorption levels. When SF concentrations exceed 2.0 w/v%, SF becomes the continuous phase, embedding the NPs and disrupting ordered assembly. Regardless of NPs, these SEM results show that an SF020 adsorption level is the most effective range for high-density NP layer coatings while using minimal amounts of SF. When it comes to forming high-density and uniform NP layers, SF002 is insufficient, SF020 level gives optimal results for all studied NPs, and SF200 is excessive. Thus, hereafter, we refer to these concentrations as ‘insufficient’, ‘optimal’, and ‘excessive’, respectively.

Figure 3b, c highlight the contrast between uniform, dense SiO2 assembly on PDMS at optimal SF concentration (i.e., SF020) and the disrupted composite-like SF–SiO2 layer with excessive SF (i.e., SF200). Excess free SF remaining in the dispersion can accumulate between NPs, leading to thicker coated films (Supplementary Fig. 16). This optimal adsorption level could vary upon the concentration and size of NPs. It should be noted that throughout this study, NP dispersions with low concentration (<10%) and nanoscale diameter (⌀<200 nm) were used.

Spin coating of various aqueous NP dispersions with optimal SF adsorption onto PTFE and PDMS surfaces results in high-density NP layers. X-ray photoelectron spectroscopy (XPS) analysis quantitatively confirms that these coatings fully obscure the underlying substrate, demonstrating effective surface coverage at the nanoscale. Figure 3d and Supplementary Fig. 17 illustrates the reduction in fluorine and silicon signals from PTFE and PDMS, respectively, after coating of NPs. XPS survey scans for each NP-coated substrate are shown in Supplementary Figs. 18, 19. These results quantitatively confirm that aqueous NP dispersions with optimal SF adsorption produced well-coated NP layers on hydrophobic substrates35.

The SF-adsorbed NP assemblies formed on hydrophobic or elastic substrates remain well-adhered and electrically function even under moderate mechanical deformation (bending radius ≥20 mm), without noticeable delamination or loss of interconnectivity (Supplementary Fig. 20). Introducing a thin elastomeric encapsulation layer further enhances the stability and reliability of these assemblies, ensuring practical applicability in flexible or bio-integrated devices (Supplementary Fig. 21).

Furthermore, the structural conformation of the SF adsorption over NP layers can be manipulated via previously reported post-processing techniques like solvent annealing (e.g., water vapor or methanol vapor annealing)36,37. Although solvent-induced crystallization may dehydrate the SF adsorption layer and alter its mechanical properties, its influence on interparticle adhesion at the optimal adsorption condition (i.e., SF020) is minimal (Supplementary Fig. 22). At this concentration, SF chains have already undergone substantial conformational rearrangement and network formation during adsorption, and further crystallization might mainly consolidate the existing adhesive interactions. Notably, crystallized SF adsorbates stabilize the interparticle packing against redispersion in water, allowing sequential coating of aqueous NP dispersions (Fig. 3e, f). Sequential spin-coating with crystallization of optimal SF-adsorbed SiO2, zinc oxide (ZnO), and indium tin oxide (ITO) NPs enables the production of a multi-layered structure with clear and distinct boundaries. In contrast, sequential coating without crystallization results in partial intermixing of the previously deposited NP layers at the interfaces (Supplementary Fig. 23).

These results demonstrate that the optimal level of SF adsorption not only maximizes the fibroin-mediated intermolecular interactions but also enables high-density NP assembly on previously non-wettable substrates via water processing. In this aqueous process, the coating behavior is jointly governed by an interfacially adsorbed SF layer that defines NP assembly and by a minor residual free-SF fraction that modulates wetting19,20. This interplay establishes an optimal adsorption window in which film coverage and uniformity are maximized. Furthermore, the polymorphism of SF offers additional benefits in processing tunability for building reliable multilayered NP heterostructures with distinct layer boundaries.

Preserved functionality of NP-based electronics at optimal SF adsorption
Optimal SF adsorption enables the water-based fabrication of high-density NP assembly, preserving the inherent electrical functionality of the original NPs38,39. In this study, three types of NPs assemblies are formed via spin coating without thermal treatments, and their electrical performance is analyzed (Fig. 4). NP coatings with excessive SF adsorption, which produce composite-like layers, are excluded from this study, as they are beyond the scope of this study. All SF-adsorbed NP assemblies are treated with methanol to crystallize the SF adsorption layer, thereby improving the device stability against ambient humidity.

Capacitors composed of SiO2 NPs with a uniform and high-density NP layer are fabricated (Fig. 4a) and characterized to assess the influence of different SF adsorption levels on their insulation and capacitance characteristics (Figs. 4b, c). As shown in current density-electric field (J–E) curves (Fig. 4b), the pure SiO2 NP capacitor and that with optimal SF adsorption exhibited comparable leakage currents and electrical breakdowns (10−7 A/cm² and 5.2 MV/cm for pure NP layers, while 10−6 A/cm2 and 4.9 MV/cm for NP with SF adsorption, respectively). On the other hand, the insufficient-SF adsorbed SiO2 NP capacitor exhibits vertical cracks across the film (see Supplementary Fig. 24a), creating electrical paths between the electrodes and leading to poor insulating performance. J–E curves for devices with varying SF adsorption levels are provided in Supplementary Fig. 25. Figure 4c shows the frequency-dependent capacitance of the NP layers. The average dielectric constants for pure crystallized SF films (thickness ~274 nm), optimal-SF adsorbed SiO2 NP capacitors (thickness ~321 nm), and pure SiO2 NP capacitors (thickness ~346 nm) were 17.4 ± 0.9, 8.21 ± 0.5, and 5.97 ± 0.4 at 10 kHz, respectively, which are comparable to other solution-processed dielectrics40,41. These results demonstrate that SF adsorption effectively facilitates the formation of high-density NP layers without compromising the intrinsic dielectric performance of the NP.

Figures 4d–f show a transparent NP conductor formed onto a PDMS strip. Optimal SF adsorption allows a continuous and closely packed ITO NPs layer on PDMS, causing the resultant film to exhibit more than double the conductivity of pure ITO NP layers and of the those formed with unoptimized/insufficient SF adsorption (Fig. 4e). These results suggest that, despite SF’s well-known dielectric nature, optimal usage of SF-adsorbate maintains physical connections between NPs, resulting in net conductivity enhancement. From a practical perspective, relying solely on zero-dimensional nanoparticles to form a strong percolation path is inefficient42. Hence, a hybrid percolation path using high-aspect-ratio (HAR) silver nanowires (Ag NWs) and ITO NPs is also explored. As shown in Fig. 4f, Supplementary Fig. 26a, introducing Ag NWs (HAR~250) beneath the ITO NP layer increases conductivity by approximately three orders of magnitude, from 50 S/m to as high as 80,000 S/m. While the hybrid NP conductors’ conductivity is 100 times lower than sputtered ITO films (~106 S/m), it was still reasonable considering contact resistance and charge carrier scattering occurring at NP junctions42,43. With appropriate encapsulation, the SF-adsorbed hybrid conductor formed on soft PDMS substrates can be mechanically stabilized, maintaining a consistent percolation network even under repeated tensile bending (up to 350 cycles at a bending radius of 20 mm; Supplementary Fig. 21b). Furthermore, compared to solution-processed conductive materials, the Ag NW/ITO NP hybrid conductor offers comparable or higher conductivity, underscoring the versatility of this approach44–46. The optical transparency is compromised by increasing Ag NWs, as the NW meshes act as optical scattering points. Thus, the trade-off between conductivity and transmittance needs to be balanced depending on application requirements. Supplementary Fig. 26b shows the transparency of the Ag NW/ITO NP hybrid conductor at different Ag NW concentrations.

Analysis of the transfer characteristics of the semiconducting NPs layer with optimal SF adsorbates is illustrated in Figs. 4g–j, Supplementary Fig. 27. Pure NP transistors and those with optimal SF adsorption exhibit similar on/off current ratios of approximately 105. On the other hand, ZnO NP layers with insufficient SF adsorbate exhibit poor surface coverage and defects (e.g., voids and cracks) (see Supplementary Fig. 24c), leading to a 70% failure rate, as shown in Supplementary Fig. 27. The subthreshold swings of both pure and SF-adsorbed ZnO NP transistors are measured to be around 250 mV/dec, showing no significant difference. In contrast, transistors with optimal-SF adsorption exhibit slightly higher mobility (~0.2 cm2/Vs) than the others (p-value~0.018). This mobility improvement is attributed to the close NP packing and dielectric screening provided by the SF adsorption layer, which enhances charge carrier transport while reducing scattering from charged impurities in the NPs42,47–49.

Methods
Materials
Water-based 10% carboxylate-modified polystyrene (PS, ⌀~100, 300, and 600 nm) nanosphere dispersions are purchased from Banglabs. Water-dispersed gold nanoparticles (AuNPs) and gold nanorods (Au NRs) are prepared with previously reported methods50,51 and as described in this “Method” section. Water-based 20% zirconium oxide (ZrO2, diameter~50 nm) dispersion is purchased from USNANO. Gold (III) chloride trihydrate (HAuCl4), sodium citrate, cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH4), silver nitrate (AgNO3), L-ascorbic acid, water-based 50% silicon dioxide (SiO2, LudoxTM-50, diameter~22 nm) colloidal, 20% indium tin oxide (ITO, diameter~18 nm), 20% zinc oxide (ZnO, diameter~40 nm) dispersions, sodium carbonate (Na2CO3), and lithium bromide (LiBr) are purchased from Sigma Aldrich. Water-based 2% silver nanowires (Ag NWs, diameter~120 nm, length~20 μm) dispersion is purchased from MSE Supplies. Lacey carbon grid for transmission electron microscopy (TEM) measurement is purchased from Ted Pella. All nanoparticles (NPs), colloidal and dispersions are used as received without further purification and diluted accordingly with deionized water (DIW, 18.2 MΩ·cm), obtained from a Milli-Q Nanopure system.

Preparation of regenerated silk fibroin (SF)
SF is prepared with a process previously described36. Briefly, Bombyx mori silk cocoons were cut and boiled in 0.02 M Na2CO3 (Sigma Aldrich) solution for 30 min to remove sericin. The dried and degummed silk was dissolved in 9.3 M LiBr (Sigma Aldrich) solution at 60°C for 1 h. The dissolved SF solution was placed into a dialysis tube (Fisherbrand, MWCO 3.5 K), followed by dialysis against DIW for 3 days with at least 5 water changes. After dialysis, the solution was purified through centrifugation at 11,400g for 20 min at 4°C. The 30 min-boiled SF prepared in this manner exhibits a molecular weight distribution of ~219–258 kDa19. The resulting SF solution was diluted to 4.0 w/v% and served as a stock solution.

Preparation of AuNPs and AuNRs
AuNPs were synthesized by boiling 100 mL of 1.0 mM HAuCl4 solution, followed by rapid injection of 10 mL of 38.8 mM sodium citrate under vigorous stirring. The reaction was maintained for 20 min until the solution turned deep red. AuNRs were synthesized using a seed-mediated growth method. The seed solution was prepared by mixing 0.4 mL of 50 mM HAuCl4 with 40 mL of 0.2 M CTAB, followed by rapid addition of 4.8 mL of ice-cold 0.01 M NaBH4 and stirring for 2 min at 25°C. For the growth solution, 40 mL of 0.2 M CTAB was combined with 0.8 mL of 4 mM AgNO3, 0.8 mL of 50 mM HAuCl4, and 0.56 mL of 0.0788 M L-ascorbic acid. Subsequently, 0.1 mL of the seed solution was added to the growth solution, and the mixture was kept at 27°C overnight to form NRs.

Preparation of SF-adsorbed NP suspensions with varying NP and SF concentration
The concentration of NPs in the water-based suspensions was appropriately adjusted according to the intended purpose, such as observing SF adsorptions in monolayer formation or coating of crack-free NP films. The concentrations of SF and NPs in the final suspensions were adjusted by mixing their stock solutions with DIW in appropriate volumetric ratios. Unless otherwise noted, all suspensions were prepared in unbuffered DIW with no added salts/ions and used at room temperature under near-neutral pH (typically pH 6.8–7.2 immediately after mixing). No wash/centrifugation step was applied to remove non-adsorbed free SF chains from the NP suspensions. Supplementary Table 1 shows an example of the mixing ratios of each stock solution and DIW used in this study.

Optical characterizations of SF-adsorbed NP suspensions and their coating results
A field emission TEM (JEOL F200, JEOL) was used to image NPs with or without SF adsorption at the acceleration voltage of 200 kV. NP suspensions were prepared according to Supplementary Table 1. For TEM, 5 μL of NP suspension was drop-cast onto lacey-carbon grids and air-dried overnight at ambient conditions, without any surface treatment or accelerated drying. Each grid was first surveyed at low magnification to locate regions where a single, close-packed particle layer spans a lacey opening; high-magnification images were then acquired to visualize the SF adsorption layer. The morphology and elemental composition of the samples were analyzed using scanning electron microscopy (SEM, EVO MA10, Zeiss). High-magnification images were acquired with a field emission SEM (FE-SEM, Sigma 300, Zeiss) equipped with an SE2 detector with a 60 µm aperture, operated at 7 kV with a working distance of 8.6 mm. To ensure electrical conductivity, the samples were mounted on aluminum stubs using conductive carbon tape and coated with approximately 13 nm of carbon using a sputter coater (Q150TES Plus, Quorum). Elemental composition analysis was performed using an energy dispersive X-ray spectrometer (EDS, Ultimax 65, Oxford Instruments) detector with Aztec 6.0 software. The elemental maps were acquired using an energy range of 10 keV, with a pixel size of 0.00082 µm and accumulating three frames per map. Digital images are taken using a digital single-lens reflex (DSLR) camera (Rebel EOS-SL1, Canon).

Correlative AFM-IR imaging of SF-adsorbed NPs assembly (nanoscale)
The surface topography and selective chemical mapping of SF-adsorbed PSNPs (Ø~500 nm) assemblies were obtained using an atomic force microscopy-infrared spectroscopy (AFM-IR, Dimension IconIR, Bruker). Prior to correlative imaging, IR spectroscopy was conducted on bare PSNPs and SF films within the 1800–800 cm−1 range to determine their characteristic IR absorption bands. Correlative imaging of NPs assemblies with various SF concentrations (i.e., 0.02 and 0.2 w/v% SF) was performed in tapping mode using a gold-coated cantilever (PR-UM-TnIR-D, Bruker). In order to enhance the IR sensitivity and suppress the substrate signal, SF-adsorbed NPs assemblies were prepared over 70 nm gold-coated glass substrates. Neither the SF films nor the SF-adsorbed PSNPs assemblies were subjected to post-crystallization treatment. Height profiles were acquired by measuring cantilever deflections over a scan area of 500 nm2, simultaneously recording IR absorbance at 1650 cm−1 to map the spatial distribution of the SF-specific Amide I band.

Correlative Raman-SEM imaging of SF-adsorbed NPs assembly (microscale)
The morphology and conformation of the microscale SF-adsorbed PS particles (Ø~20 µm) assemblies were investigated using the Fe-SEM (Sigma 300, Zeiss) with an in situ confocal Raman RISE setup. This setup enables confocal Raman imaging of the same sample positions from which the SEM images are taken52. Samples were mounted on aluminum stubs using conductive carbon tape and imaged as is with no additional coating. SEM images were acquired under high vacuum at low acceleration voltages EHT=0.7 kV, with a secondary InLens detector, and working distances set to WD=4-4.4 mm. Raman spectra were acquired using a Witec WRL-532-E-100-TP laser (λexcitation= 532 nm, P=75 mW) equipped with a UHTS300 VIS imaging spectrograph (600 g/mm grating), a PE cooled CCD, and a 3D piezo scan stage (250 µm x 250 µm x 250 µm). The laser was focused through a Zeiss LD EC Epiplan-Neofluar Vac Dic 100x objective (WD=4 mm, NA=0.75). Spectra were acquired at laser power P=5 mW, integration time t=1 s, and accumulations N=30, while working at room temperature. Acquired Raman spectra were further processed using the Witec Control 6.2 software to perform cosmic ray removal, background subtraction, and smoothing using the Savitzky-Golay method. Corresponding optical micrographs were acquired using a DFK-33GX178 Imaging Source camera.

Hydrodynamic characterization of SF-adsorbed NP suspensions
Dynamic light scattering (DLS) and zeta potential measurements were performed using a Zetasizer Pro (Malvern) to determine the hydrodynamic radius of the particles and assess the stability of NP suspensions at varying SF concentrations (ranging from 0 w/v% to 2.0 w/v%). For reference, the zeta potential of a 5.0 w/v% aqueous silk solution was also measured from three different samples and found to be −3.17 ± 0.48 (s.d.) mV. To maintain a pH, 100 µL of 1X phosphate-buffered saline (PBS, pH 7.4) was added to each tested dispersion.

Adhesion characterization between SF-adsorbed colloidal probes and hydrophobic substrates
A bio-AFM (JPK Nanowizard, Bruker), along with a diamond-like carbon cantilever (Biosphere B300-FM, nanotools) or a colloidal cantilever (CP-PNPS-PS-A-5, sQube), was used to measure the interaction force between the NP layer and the cantilever. The cantilever approached the NP layer and was held for varying seconds from 0.02 s to 120 s after the contact, then retracted. The compressing force was set to 1 nN for characterizing time-dependent adhesion behavior; otherwise set to 10 nN. The adhesion force and energy dissipation between the NP layer and the cantilever were extracted from the force-distance (FD) curves using JPK SPM Data processing software. To characterize the changes in elastic modulus near the interface region, area-corrected fitting, tailored to the probe geometry, was performed on approach FD curves using the user-defined fitting function of OriginPro 2019 software. Similarly, worm-like chain (WLC) polymer extension model fitting was conducted on the observed elongation peaks in retracting FD curves in the same manner.

Optical and surface chemical characterization of SF-adsorbed NP coatings
Visual inspection for NP coatings (e.g., surface coverage and cracks, etc.) was performed using an optical microscope (OM) (Eclipse LV100, Nikon) with 20x and 100x objective lenses and top-view SEM observation. The thickness of NP coatings was measured using cross-sectional view SEM observation, cross-checked with a spectroscopic ellipsometer (RC2, J.A. Woollam). X-ray photoelectron spectroscopy (XPS) (K-Alpha plus, Thermo Fisher Scientific) was used to measure the atomic composition of the NP coating and the substrate.

Preparation of SF-adsorbed NP coatings for electronic devices
Water-based dispersions of 4% SiO2, 10% ITO, 0.5~2% Ag NWs, and 10% ZnO with varying SF concentrations (0, 0.02, and 0.2 w/v%) were prepared for device fabrication. UV ozone-treated substrates were used for coating dispersions without silk, while pristine substrates were used for those with silk. For SiO2 NP-based capacitors, a SiO2 colloidal solution was spin-coated at 2000 rpm for 60 s onto ITO/glass substrates. Circular electrodes (⌀~300 µm) were thermally deposited on the SiO2 NP layer coating. An 8 w/v% silk solution was spin-coated at 2000 rpm onto ITO/glass substrates for the comparison sample. For ITO/Ag NWs-based transparent conductors, Ag NWs dispersions were spin-coated at 4000 rpm for 10 s onto clean polydimethylsiloxane (PDMS) strips, followed by spin-coating ITO dispersions at 2000 rpm for 60 s over the Ag NWs layer. For bottom-gated ZnO NP-based field-effect transistors (FETs), a ZnO dispersion was spin-coated at 2000 rpm for 60 s onto a 250 nm-thick SiO2/silicon substrate (As-doped, 0.001~0.005 Ohm-cm). Aluminum electrode pairs (gap ~100 µm, width ~800 µm) were thermally deposited on the ZnO NP layer. All NP layers were crystallized by spin-coating 50 µL of pure methanol at 2000 rpm for 60 s.

Characterization of SF-adsorbed NP electronic devices
The electrical properties of the aqueous NP-based devices were analyzed using a parameter analyzer (4200A-SCS, Keithley). The current density-electric field (J-E) curves for the SiO2 NP layer and silk film (thickness ~700 nm) were obtained by linearly sweeping the voltage from −2 to 200 V across top-bottom aluminum electrodes. The measured current and voltage were converted to current density and electric field using the film thickness and electrode area. Capacitance per unit area was calculated by measuring the dielectric layer’s capacitance over 1 kHz to 1 MHz and dividing it by the electrode area. The dielectric constant was determined from the calculated capacitance per unit area, the vacuum permittivity, and the measured film thickness. The sheet resistance of ITO/Ag NWs-based transparent conductors was measured using a four-point probe (FPP-5000, Miller) and then converted to conductivity using the measured film thickness. The spectral transmittance of these conductors was measured using a custom-built microscope spectrometer setup (USB2000, Ocean Optics). The transfer characteristics of ZnO-based transistors were inspected via three-terminal measurements where drain electrodes were grounded while the source-gate voltage (VGS) and source-drain voltage (VDS) were manipulated. Electron mobility and subthreshold swing of the ZnO NP layer were extracted from the forward sweep transfer curves.

Statistical analysis of the acquired data
At least three data points were collected for each measurement in this study. Mean and standard deviation were calculated from these data points and are presented as indicated in the figure captions. Analysis of variance (ANOVA) and paired t-tests with an alpha value of 0.05 were performed for mean comparisons using JMP Pro 17 software. No data were excluded from the analyses, and all experiments were conducted without randomization. The statistical significance is denoted as not significant (ns), *P<0.05, **P<0.01, and ***P<0.001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present a work in which they demonstrate that silk fibroin (SF) is suitable to form high-density layers on nanoparticle (NP) surfaces. As SF is water-soluble, this “coating” of NPs happens in an aqueous dispersion, which is advantageous. Various types of NPs are used, most of all polystyrene spheres and silica NPs. For the characterization of the modified NPs a zoo of experimental tools is applied and gives very reliable information.\nIt is claimed that the given approach will allow to produce bio-nano interfaces for various applications.\nIn the conclusions it is stated that an optimal SF concentration of 0.2 w/v% was found for the discussed applications. Lazy readers, just reading abstract and conclusions could conclude that this is true for any NPs, however, the optimum SF concentration should depend on the size, shape and surface characteristics, as it is always the case.\nExcept for this given optimum SF concentration (maybe my personal pessimistic view of possible ignorant readers), this is an excellent manuscript and deserves to be published in a leading journal.", "reviewer_2": "T. Kim and colleagues’ study, “Enabling Water-based, High-Density Nanoparticles Assembly by using Silk Fibroin as an Adsorbate,” investigates a water-based fabrication strategy for producing high-density nanoparticle (NP) layers by exploiting silk fibroin (SF) as a natural amphiphilic adsorbate that improves wetting and packing on hydrophobic substrates. The authors support their conclusions with an impressive array of physico-chemical and electrical measurements—TEM/SEM, AFM-IR, DLS and ζ-potential, AFM force–distance spectroscopy, XPS, ellipsometry, correlative Raman-SEM, and device-level electrical tests—and the experimental descriptions are clear and detailed.\nThis work is significant for bio-integrated interfaces, solution-processed electronics, and nanomaterial assembly, and it will appeal to the Nature Communications readership interested in water-based nanofabrication and biotic–abiotic interface technologies.\nI recommend consideration for publication after the authors address the following comments.\n1. In the “Concentration-dependent adsorption of SF onto NPs” section, it is not clear how the nanoparticle monolayer in Fig. 1d was prepared. The Methods state only that NP dispersions were drop-cast and dried overnight on lacey-carbon TEM grids. Under such conditions, nanoparticles typically self-assemble vertically into 3-D opal-like multilayers. Please clarify whether the monolayer was obtained through surface treatment, dilution control, or shortened drying, or whether the image shows the top surface of a thicker stack.\n2. In Fig. 1f, the hydrodynamic radius increases with SF concentration up to about 0.4 w/v % but slightly decreases at higher concentrations, while the ζ-potential changes little. A short discussion of this non-monotonic trend would help readers; possible reasons include screening of electrostatic repulsion, partial compaction of SF chains, or apparent boundary shifts caused by free SF molecules.\n3. In Fig. 1g, the AFM force-spectroscopy results compare pristine and SF-adsorbed NPs. It would improve clarity to include adhesion-force data as a function of SF concentration (e.g., 0.02 %, 0.2 %, 2 %) to illustrate the transition among “insufficient,” “optimal,” and “excessive” adsorption regimes. Supplementary Fig. 9 shows only pristine and 2 %, so additional data would be valuable.\n4. In the “Wetting improvement and high-density NP coatings enabled by optimal SF adsorption” section, the authors convincingly demonstrate dense NP coating on hydrophobic substrates (PDMS, PTFE). For applications in bio-integrated or flexible devices, please discuss whether the SF-bound NP layer remains mechanically and electrically stable under bending. Since Fig. 4d later presents a flexible device, a comment on time-dependent adhesion retention and mechanical durability would strengthen the discussion.\n5. In Fig. 3b and 3c, the NP layer at higher SF concentration (2 wt %) appears slightly thicker. A brief discussion of film-thickness dependence on SF concentration at identical spin-coating speeds—supported by SEM or ellipsometry data—would clarify whether this difference arises from excess SF matrix or modified wetting.\n6. In Fig. 3e, solvent-annealing experiments show that SF crystallization improves multilayer stability. For a single-layer film, however, does crystallized SF exhibit a different adhesion force than amorphous SF? Because SF with higher β-sheet content may have greater stiffness and lower chain mobility, a cautious discussion of how such structural differences could influence interfacial adhesion would provide deeper mechanical insight.\n7. In the “Preserved functionality of NP-based electronics at optimal SF adsorption” section, the authors demonstrate several devices using SF-adsorbed NP layers. It would be valuable to discuss mechanical and electrical stability during substrate bending, especially for the hybrid Ag NW/ITO electrode on PDMS (Fig. 4d). Since Ag NWs bridge the inorganic ITO and soft PDMS, commenting on how this network maintains conductivity and adhesion under strain would emphasize the practical importance of the approach.\n8. In Fig. 4, the notations SF020, SF002, and SF film appear in several graphs. These abbreviations are defined only in Supplementary Table 1. They should be clearly defined in the main text (e.g., SF020 = 0.2 wt % SF, SF002 = 0.02 wt % SF) so readers need not consult the Supplementary Information. Because dielectric thickness strongly affects capacitor performance, please also provide approximate film thicknesses for these conditions.\n9. The Supplementary Information presents LSPR spectra of Au NPs and Au NRs on PDMS, but the optical structure of these plasmonic samples is not described—e.g., film thickness, particle density, or whether a monolayer or thicker layer was used. Since plasmonic response depends on particle arrangement, a concise description of the coating morphology and thickness would make the optical data more meaningful.\nOverall, this manuscript provides a thorough and convincing study of silk-fibroin-mediated nanoparticle assembly using fully water-based processes. The data are comprehensive and of high quality, and the work establishes SF as an enabling material for environmentally friendly nanofabrication. Addressing the points above—particularly clarification of sample preparation, film-thickness and concentration dependencies, and mechanical stability of flexible devices—would further strengthen the paper and broaden its impact.", "reviewer_3": "This manuscript describes the use of silk fibroin to assist with the formation of dense nanoparticle layers. The authors show that silk fibroin can be non-specifically adsorbed onto the surface of nanoparticles, and that spincoating a nanoparticle/silk fibroin solution onto silicon yields a dense nanoparticle layer. The authors pinpoint a specific concentration range of silk fibroin that yields best results in terms of conductivity and device performance (shown by a model nanoparticle-based capacitor). The experiments are comprehensive, leveraging a variety of techniques for molecular-scale characterization (e.g. of adhesion force, morphology, surface charge, device performance metrics, etc). The data analysis is very competent, and I commend the authors for presenting a very clear manuscript.\nOne of my major critique concerns the novelty and significance of the work. The adsorption of silk fibroin to surfaces has been previously reported by Kaplan and colleagues as far back as 2005 (https://doi.org/10.1021/la051862m, https://doi.org/10.1016/j.jconrel.2007.06.006, https://doi.org/10.1002/adfm.200901774 - this last one even reports coating nanoplatelets) and by Zha and colleagues (https://pubs.rsc.org/en/content/articlepdf/2018/bm/c8bm01186a, https://doi.org/10.1021/acsbiomaterials.3c01042, https://doi.org/10.1021/acsami.4c07528). The prior literature shows many relevant aspects, such as the effect of pH, presence of ions, secondary structure change upon adsorption, and adhesion (also measured by AFM force spectroscopy). However, there is insufficient discussion in the manuscript of the submitted work in context of these prior works.\nThe ability of silk fibroin to adsorb to surfaces, and that absorption amount can be \"tuned\" by changing silk fibroin concentration, is unsurprising, as this is typical macromolecular adsorption behavior. I think the novelty of this manuscript is the identification of a silk fibroin concentration that can give just enough \"stick\" to the nanoparticles to assist with forming a dense, conductive layer. However, I would like a better discussion in the manuscript about how silk fibroin compares to other (potentially cheaper) molecules that can modify wettability, surface tension, and adhesion (such as surfactants, block copolymers, amphiphilic, non-amphiphilic polypeptides, oligo/polysaccharides, etc). Is there something special about the silk fibroin sequence that allows to behave this way? How does the use of silk fibroin stack up against other methods of improving nanoparticle wettability? This discuss is rather vague in the manuscript.\nThe claim that these devices can perform well in a biological setting is perhaps a bit of a stretch. Silk fibroin will swell (increasing the distance between nanoparticles) and be degraded over time if it is in contact with biological fluids. The electronic properties will certainly change over time. I don't see any mention of using an alcohol or annealing post-treatment of the films, which means that the silk fibroin will likely dissolve in an aqueous environment.\nI also have a few technical questions. Can the AFM IR data be deconvoluted to get information about secondary structure composition? The signal at 1650 cm-1 is a random coil/alpha helix signal, but I would expect there to be more beta-sheets in silk fibroin, particularly if this material is expected to not dissolve in aqueous environments (see https://doi.org/10.1021/acsami.4c07528 for similar AFM-IR analysis)\nSome parameters critical to macromolecular adsorption are not included in the manuscript, such as molecular weight (and distribution) of the silk fibroin, solution pH (whether it was buffered or not), and ion content. Without this information, reproducing the results (e.g. using a different silk fibroin source/batch, in a different lab, etc) will likely be very difficult.\nThe coating process is a bit confusing to me. Was non-adsorbed silk fibroin washed away (for example, but spinning down the nanoparticles then resuspending in clean water) before spin coating? There will obviously be excess silk fibroin between particles in the case of higher silk concentration if there's no wash step. Incorporating a wash step may mean that the coating process is less sensitive to the concentration of the adsorption solution. Furthermore, if the non-adsorbed silk fibroin is still present in the spin coating step, the surface tension may be different with different concentrations, which may affect the outcomes of the spin coating.\nThe adhesion measurements in AFM were performed using a particle on the AFM tip and a flat hydrophobic substrate. Is this an acceptable model for the suspended nanoparticle system, where both surfaces have substantial curvature? I think this model needs to be justified by at least showing that silk fibroin coating thicknesses and secondary structures are similar on the flat surface vs the curved nanoparticle surface."}, "author_response": "We sincerely appreciate the reviewers’ constructive and thoughtful comments, which have been\ninvaluable in improving our manuscript.\nFor clarity, the reviewers’ comments are presented in blue text, followed by our point-by-point\nresponses. Corresponding revisions in the main manuscript and the supplementary information are\nhighlighted with a yellow background.\n● Pages 2: Response to Reviewer #1\n● Pages 3–17: Response to Reviewer #2\n● Pages 18–29: Response to Reviewer #3\nGeneral comment:\nThe authors present a work in which they demonstrate that silk fibroin (SF) is suitable to form\nhigh-density layers on nanoparticle (NP) surfaces. As SF is water-soluble, this “coating” of NPs\nhappens in an aqueous dispersion, which is advantageous. Various types of NPs are used, most of\nall polystyrene spheres and silica NPs. For the characterization of the modified NPs a zoo of\nexperimental tools is applied and gives very reliable information.\nIt is claimed that the given approach will allow to produce bio-nano interfaces for various\napplications. In the conclusions it is stated that an optimal SF concentration of 0.2 w/v% was found\nfor the discussed applications. Lazy readers, just reading abstract and conclusions could conclude\nthat this is true for any NPs, however, the optimum SF concentration should depend on the size,\nshape and surface characteristics, as it is always the case.\nExcept for this given optimum SF concentration (maybe my personal pessimistic view of possible\nignorant readers), this is an excellent manuscript and deserves to be published in a leading journal.\nResponse:\nWe sincerely appreciate the reviewer’s positive and encouraging comments on our work, and we\nare grateful for the reviewer’s recommendation for publication.\nGeneral comment:\nT. Kim and colleagues’ study, “Enabling Water-based, High-Density Nanoparticles Assembly by\nusing Silk Fibroin as an Adsorbate,” investigates a water-based fabrication strategy for producing\nhigh-density nanoparticle (NP) layers by exploiting silk fibroin (SF) as a natural amphiphilic\nadsorbate that improves wetting and packing on hydrophobic substrates. The authors support their\nconclusions with an impressive array of physico-chemical and electrical measurements—\nTEM/SEM, AFM-IR, DLS and ζ-potential, AFM force–distance spectroscopy, XPS, ellipsometry,\ncorrelative Raman-SEM, and device-level electrical tests—and the experimental descriptions are\nclear and detailed.\nThis work is significant for bio-integrated interfaces, solution-processed electronics, and\nnanomaterial assembly, and it will appeal to the Nature Communications readership interested in\nwater-based nanofabrication and biotic–abiotic interface technologies.\nI recommend consideration for publication after the authors address the following comments.\nResponse:\nWe sincerely appreciate the reviewer’s positive and encouraging assessment of our work. We are\ngrateful for the thoughtful and detailed comments, which have helped us substantially strengthen\nthe clarity, rigor, and completeness of the manuscript. In response, we have expanded our analysis\nand revised the text accordingly. We believe the following revisions address the reviewer’s\ncomments with care and further reinforce the significance and robustness of our water-based SF-\nadsorbate nanofabrication strategy. We sincerely thank the reviewer again for the constructive\nfeedback and for recognizing the potential impact of our work.\nIn the “Concentration-dependent adsorption of SF onto NPs” section, it is not clear how the\nnanoparticle monolayer in Fig. 1d was prepared. The Methods state only that NP dispersions were\ndrop-cast and dried overnight on lacey-carbon TEM grids. Under such conditions, nanoparticles\ntypically self-assemble vertically into 3-D opal-like multilayers. Please clarify whether the\nmonolayer was obtained through surface treatment, dilution control, or shortened drying, or\nwhether the image shows the top surface of a thicker stack.\nResponse:\nWe appreciate the reviewer’s point and agree that our initial Methods lacked sufficient detail on\nTEM sample preparation and image selection.\nThe TEM panels in Fig. 1d were acquired from monolayer domains that naturally form\nacross lacey-grid openings during drying, where a single particle layer is exposed at the top surface.\nDispersions were prepared following Supplementary Table 1. For TEM, 5 μL of each dispersion\nwas drop-cast onto lacey-carbon grids and air-dried overnight, without surface treatment or\naccelerated drying. Indeed, under these conditions, both monolayer domains and thicker vertical\nstacks coexist across a grid. Before high-magnification imaging, we surveyed each grid at low\nmagnification to locate areas exhibiting a single, close-packed layer spanning a lacey opening; we\nthen focused at the outer rim of these monolayer domains to better visualize the SF overlayer. At\n2.0 w/v% SF, thicker stacks occurred more frequently; in such cases, we prepared 2–3 additional\ngrids and repeated the survey to secure monolayer fields of view. We have revised the Methods to\ndescribe these procedures in detail.\nCorresponding revisions:\nPage 9, line 357 in the main manuscript\nA field emission TEM (JEOL F200, JEOL) was used to image NPs with or without SF adsorption\nat the acceleration voltage of 200 kV. NP suspensions were prepared according to Supplementary\nTable 1. For TEM, 5 μL of NP suspension was drop-cast onto lacey-carbon grids and air-dried\novernight at ambient conditions, without any surface treatment or accelerated drying. Each grid\nwas first surveyed at low magnification to locate regions where a single, close-packed particle\nlayer spans a lacey opening; high-magnification images were then acquired to visualize the SF\nadsorption layer.\nIn Fig. 1f, the hydrodynamic radius increases with SF concentration up to about 0.4 w/v %\nbut slightly decreases at higher concentrations, while the ζ-potential changes little. A short\ndiscussion of this non-monotonic trend would help readers; possible reasons include screening of\nelectrostatic repulsion, partial compaction of SF chains, or apparent boundary shifts caused by free SF\nmolecules.\nResponse:\nWe appreciate the reviewer’s insightful comment.\nAs pointed out, Fig. 1f could visually suggest a mild downturn; however, this is not\nsupported by statistics. Specifically, the hydrodynamic radii of PS across 0.2, 0.4, 1.0, and 2.0 w/v%\nSF (n=3 per group) are 109.3±2.9, 110.7±2.5, 110.1±1.9, 109.9±1.8 nm (mean ± s.d.), and a one-\nway ANOVA analysis among these groups indicates no significant difference (p ~ 0.89). We\ntherefore interpret the hydrodynamic radius of PS beyond 0.4 w/v% of SF adsorption trend as a\nplateau within experimental variability rather than a true decrease. Consistently, the zeta-potential\nchanges little and gradually approaches that of the SF solution at pH 7.4, supporting continued SF\ncoverage rather than desorption. Across other nanoparticle types (see Supplementary Fig. 7), the\nhydrodynamic radius of NPs increases or levels off with SF; we did not observe a systematic\ndownturn.\nTo clarify these points, a brief sentence has been added to the manuscript and Fig. 1f has\nbeen updated to display statistical information.\nCorresponding revisions:\nPage 4, line 125 in the main manuscript\nAs the SF content in aqueous PSNP dispersion (⌀ ~ 100 nm) increases, the hydrodynamic radius\nrises and plateaus by ~0.2 w/v%, and beyond this point, no significant differences are observed (p-\nvalue ~ 0.89).\nPage 18, Figure 1f in the main manuscript\nIn Fig. 1g, the AFM force-spectroscopy results compare pristine and SF-adsorbed NPs. It would\nimprove clarity to include adhesion-force data as a function of SF concentration (e.g., 0.02 %,\n0.2 %, 2 %) to illustrate the transition among “insufficient,” “optimal,” and “excessive” adsorption\nregimes. Supplementary Fig. 9 shows only pristine and 2 %, so additional data would be valuable.\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion.\nAs recommended, we performed additional AFM force–distance measurements to quantify\nhow adhesion force varies with SF concentration. Using similar experimental settings as in the\noriginal Fig. 1g (i.e., 300 nm polystyrene NPs and a 300 nm-radius diamond-like carbon colloidal\nprobe), we measured adhesion force between the colloidal probe and pristine particles (0% w/v\nSF) and those with three SF adsorption levels: 0.02, 0.2, and 2.0 w/v%. Please note that in these\nnew measurements, the probe was retracted immediately upon reaching the force setpoint, without\nan intentional dwell period, in order to minimize potential time-dependent changes in adhesion.\nThe results show a clear non-monotonic trend. Adhesion increases only modestly at 0.02%\nSF (“insufficient adsorption”), reaches a pronounced maximum at 0.2% SF (“optimal adsorption”),\nand decreases again at 2.0% SF (“excessive adsorption”), approaching the pristine level. Statistical\nanalysis (one-way ANOVA followed by pairwise Student’s t-tests) confirms that only the 0.2%\ngroup exhibits a significant increase in adhesion compared to all other concentrations, while the\n0%, 0.02%, and 2.0% groups are statistically indistinguishable.\nNotably, this concentration-dependent adhesion behavior is fully consistent with our AFM-\nbased intermolecular interaction analysis (Fig. 2), where the 0.2% SF condition likewise yields the\nlargest adhesion force and energy dissipation. These new data therefore not only directly address\nthe reviewer’s comment but also quantitatively establish the transition between insufficient,\noptimal, and excessive SF adsorption regimes.\nWe have revised Fig. 1g in the main manuscript to include this full concentration-\ndependent analysis and added discussion regarding this.\nCorresponding revisions:\nPage 4, line 136 in the main manuscript\nThe SF adsorption-induced change in NP surface energy (Fig. 1g and Supplementary Fig. 9) is\nconfirmed by AFM force spectroscopy. The adhesion force between a diamond-like carbon\ncolloidal probe and SF-adsorbed NPs exhibits a clear non-monotonic dependence on SF\nconcentration: adhesion increases from the pristine (~14.4 nN) and SF002 conditions (~22.3 nN)\n6\nto a pronounced maximum at SF020 (~32.3 nN) and then decreases again at SF200 (~21.8 nN).\nThis result identifies that there is an optimal adsorption window maximizing the particle-to-\nparticle interaction.\nPage 18, Figure 1g in the main manuscript\nIn the “Wetting improvement and high-density NP coatings enabled by optimal SF adsorption”\nsection, the authors convincingly demonstrate dense NP coating on hydrophobic substrates (PDMS,\nPTFE). For applications in bio-integrated or flexible devices, please discuss whether the SF-bound\nNP layer remains mechanically and electrically stable under bending. Since Fig. 4d later presents\na flexible device, a comment on time-dependent adhesion retention and mechanical durability\nwould strengthen the discussion.\nResponse:\nWe thank the reviewer for raising this important point regarding the mechanical stability of SF-\nadsorbed NP layers. For bending tests, we focused on the optimized SF (i.e., SF020) Ag NW/ITO\nNP hybrid conductor on PDMS; the other proof devices (SiO capacitors and ZnO transistors)\n2\nwere fabricated on rigid ITO-glass or doped-Si substrates and are outside the scope of bending\ncharacterization. Here, R denotes the resistance in the flat state, and r and r are the bending radii\n0 c t\nunder inner (compressive) and outer (tensile) bending, respectively.\nAs shown in newly added Supplementary Fig. 20, the hybrid conductor exhibits\nasymmetric behavior under compression versus tension. Under compressive bending the relative\nresistance decreases modestly (ΔR/R ~ −0.63 at r = 30 mm; ~ −0.71 at r = 10 mm), consistent\n0 c c\nwith enhanced Ag NW–Ag NW/NP contacts and previous report (Appl. Phys. Lett. 110, 221906\n(2017)). In contrast, under tensile bending the resistance increases progressively (ΔR/R ~ 1.18 at\n0\nr = 30 mm; ~ 3.80 at r = 10 mm), attributable to tensile micro-crack formation within the brittle\nt t\nITO NP network. These data indicate that the SF-adsorbed NP network remains electrically\nfunctional and well adhered to PDMS at moderate bending radii (~20 mm), whereas more severe\ntensile bending degrades network interconnectivity.\nBecause exposed NP layers can be vulnerable to surface wear and repeated strain, we\nfurther applied a thin PDMS over-layer. Encapsulation markedly improved cyclic durability:\nΔR/R remained essentially constant up to 350 cycles, while the unencapsulated film showed a\n0\ngradual rise after ~100 cycles (see newly added Supplementary Fig. 21). Together, these results\nshow that SF-assisted NP coatings are mechanically compliant and electrically resilient for\nflexible/bio-integrated electronics, especially when combined with conventional encapsulation.\nWe have added Supplementary Figs. 20-21 and corresponding text in the main manuscript\nto document these findings.\nCorresponding revisions:\nPage 6, line 214 in the main manuscript\nand electrically function even under moderate mechanical deformation (bending radius ≥ 20 mm),\nwithout noticeable delamination or loss of interconnection (Supplementary Fig. 20). Introducing a\nthin elastomeric encapsulation layer further enhances the stability and reliability of these\nassemblies, ensuring practical applicability in flexible or bio-integrated devices (Supplementary\nFig. 21).\nPage 26 in the Supplementary information\nSupplementary Figure 20. Electrical performance of SF-adsorbed Ag NW/ITO NP\nconductors formed on a PDMS substrate under mechanical deformations.\na, A schematic of compressive (positive) bending with bending radius of r . b, Relative resistance\nc\nchanges of hybrid conductor under varying compressive r . c, A schematic of tensile (negative)\nc\nbending with bending radius of r. d, Relative resistance changes of hybrid conductor under\nt\nvarying tensile r. In b and d, data points and error bars indicate mean ± standard deviation (SD)\nt\n(N=10).\n9\npage 27 in the Supplementary information\nSupplementary Figure 21. Cyclic-bending stability of SF-adsorbed Ag NW/ITO NP\nconductors formed on a PDMS substrate.\na, Resistance of Ag NW/ITO NP hybrid conductors during repeated banding cycles at bending\nradius (r) of 20 mm. b, Relative resistance changes of Ag NW/ITO NP hybrid conductor with and\nt\nwithout thin PDMS encapsulation over 350 cycles. c. SEM images showing top view of\nunencapsulated Ag NW/ITO NP hybrid conductors after 100 and 200 cycles. Yellow arrows\nindicate cracks on the ITO NP layer. d, SEM images showing cross section of encapsulated Ag\nNW/ITO NP hybrid conductors after 100 and 200 cycles.\nIn Fig. 3b and 3c, the NP layer at higher SF concentration (2 wt %) appears slightly thicker.\nA brief discussion of film-thickness dependence on SF concentration at identical spin-coating\nspeeds—supported by SEM or ellipsometry data—would clarify whether this difference arises\nfrom excess SF matrix or modified wetting.\nResponse:\nWe thank the reviewer for the insightful comment.\nTo clarify the possible influence of SF concentration on film thickness, we measured the\nthickness of SiO NP layers spin-coated (2000 rpm, 60 s) from aqueous dispersions containing\n2\ndifferent SF concentrations (0.02, 0.2, and 2.0 w/v%) on PDMS-coated glass substrates (~1 cm2).\nThe ellipsometric thicknesses at the center region were 242.5 ± 3.7, 244.5 ± 3.7, and 285.6 ± 5.9\nnm, respectively (N = 5).\nPairwise t-tests reveal no statistical difference in thickness between 0.02 % and 0.2 % SF\ndispersions (p = 0.51), whereas the 2.0 % group exhibits a significantly thicker film (p < 0.0001).\nThis increase originates from the accumulation of free (non-adsorbed) SF molecules that remain\nin the dispersion at higher concentrations, forming an additional matrix phase among the NPs.\nAlthough wetting improvement above 0.2 % SF allows uniform coating on hydrophobic substrates\n(see Supplementary Figs. 14–15), such wetting enhancement alone cannot account for the\npronounced thickness increase at 2.0 %. Furthermore, the viscosity and surface tension of SF\nsolutions below 2 w/v % are known to be comparable to those of pure water (Macromol. Biosci.\n8, 1006–1018 (2008)), indicating that modified wetting or rheological effects are unlikely to be\nthe primary cause. Therefore, the observed thickness increase at 2.0 % SF mainly results from the\npresence of excess SF matrix rather than changes in wetting behavior.\nWe revised the main manuscript to clarify the correlation between film thickness and\nexcessive SF in NP dispersion, and added a Supplementary Fig. 16 to support this claim.\nCorresponding revisions:\nPage 5, line 202 in the main manuscript\nFig. 3b and 3c highlight the contrast between uniform, dense SiO2 NP assembly on PDMS at\noptimal SF concentration (i.e., SF020) and the disrupted composite-like SF-SiO2 layer with\nexcessive SF (i.e., SF200). Excess free SF remaining in the dispersion can accumulate between\nNPs, leading to thicker coated films (Supplementary Fig. 16).\nSupplementary Figure 16. Spectroscopic ellipsometry analysis of spin-coated NP layers with\ndifferent SF concentrations (0.02, 0.2, and 2.0 w/v% SF).\na, thickness of NP layers coated from 4% SiO dispersion with varying SF concentrations. b,\n2\nrefractive index at 633 nm of NP layers coated from 4% SiO dispersion with varying SF\n2\nconcentrations.\nIn Fig. 3e, solvent-annealing experiments show that SF crystallization improves multilayer\nstability. For a single-layer film, however, does crystallized SF exhibit a different adhesion force\nthan amorphous SF? Because SF with higher β-sheet content may have greater stiffness and lower\nchain mobility, a cautious discussion of how such structural differences could influence interfacial\nadhesion would provide deeper mechanical insight.\nResponse:\nWe are very grateful to the reviewer for this insightful question, which allows for a deeper\nmechanical interpretation of our data.\nTo directly address the reviewer’s point in the context of the practically relevant case used\nin Fig. 3, we examined how crystallization of the SF adsorption layer influences adhesion for close-\npacked NP monolayers prepared at the optimal SF concentration (SF020). After forming SF020-\nmediated PS NP monolayers, we induced β-sheet–rich crystallization by methanol treatment and\nthen measured the adhesion between a diamond-like carbon colloidal probe and the NP layer,\nfollowing the same AFM protocol as in Fig. 1g.\nThe mean adhesion force for SF020 was essentially unchanged before (Mean ± s.d.:\n~32.3±11.6 nN) and after methanol treatment (~29.0±5.4 nN) (p-value ~ 0.49, one-way ANOVA),\nindicating that crystallization does not significantly alter the average interfacial adhesion under\nthis optimal adsorption condition (see newly added Supplementary Fig. 22). However, the spread\nof the adhesion values decreased after crystallization, suggesting that the SF020 layer becomes\nmechanically more reproducible and reliable upon β-sheet formation.\nWe interpret these findings as follows. At the SF020 condition, SF chains already undergo\nsubstantial conformational rearrangement and network formation during the aqueous adsorption\nand NP-assembly steps, as discussed in the main text. As a result, methanol-induced crystallization\nprimarily consolidates this pre-organized SF network—by dehydrating and stiffening the layer—\nrather than further increasing the interfacial work of adhesion. In other words, structural\ndifferences between amorphous and crystalline SF at the optimal adsorption level mainly manifest\nas enhanced mechanical stability and reduced variability, rather than a change in the mean\nadhesion force. We have added this discussion to the revised manuscript in the Fig. 3 section and\nSupplementary fig. 22.\nCorresponding revisions:\nPage 6, line 222 in the main manuscript\nmechanical properties, its influence on interparticle adhesion at the optimal adsorption condition\n(i.e., SF020) is minimal (Supplementary Fig. 22). At this concentration, SF chains have already\nundergone substantial conformational rearrangement and network formation during adsorption,\nand further crystallization might mainly consolidate the existing adhesive interactions.\nPage 28 in the Supplementary information\nSupplementary Figure 22. Effect of crystallization on the adhesion of SF020-mediated NP\nmonolayers.\na, Schematic illustration of the AFM force–distance measurement performed on close-packed PS\nNP monolayers with amorphous and crystalline SF adsorption layers. b, Adhesion force\ncomparison for PS NP monolayers with optimal SF adsorption before and after methanol-induced\ncrystallization.\nIn the “Preserved functionality of NP-based electronics at optimal SF adsorption” section, the\nauthors demonstrate several devices using SF-adsorbed NP layers. It would be valuable to discuss\nmechanical and electrical stability during substrate bending, especially for the hybrid Ag NW/ITO\nelectrode on PDMS (Fig. 4d). Since Ag NWs bridge the inorganic ITO and soft PDMS,\ncommenting on how this network maintains conductivity and adhesion under strain would\nemphasize the practical importance of the approach.\nResponse:\nWe appreciate the reviewer for this constructive comment highlighting the importance of\nevaluating mechanical and electrical stability during bending, particularly for the hybrid Ag\nNW/ITO NP electrode on PDMS (Fig. 4d).\nAs discussed in our response to Comment 4 (page 7 of this letter), the SF-adsorbed Ag\nNW/ITO hybrid electrode exhibits asymmetric electrical responses depending on the bending\ndirection: under tensile (outer) bending, the resistance increases due to micro-crack formation in\nthe brittle ITO NP layer, whereas under compressive (inner) bending, the resistance slightly\ndecreases owing to enhanced percolation between adjacent Ag NWs and NPs. We also\nacknowledge that uncovered NP assemblies can undergo irreversible damage, such as cracking\nand local delamination, under repeated mechanical deformation.\nIn practical applications, however, such NP assemblies are typically encapsulated within a\nthin elastomeric over-layer. This encapsulation effectively enhances the mechanical stability of\nthe hybrid electrode, maintaining nearly constant relative resistance (ΔR/R ) even after more than\n0\n350 bending cycles (Supplementary Fig. 21).\nAccordingly, we have expanded the discussion in the main manuscript to describe the\ncyclic bending test results and to highlight how encapsulation mitigates mechanical fatigue and\npreserves adhesion. We hope that these revisions emphasize the practical robustness of the SF-\nadsorbed hybrid electrode architecture for flexible and bio-integrated electronic applications.\nCorresponding revisions:\nPage 7, line 278 in the main manuscript\nWith appropriate encapsulation, the SF-adsorbed hybrid conductor formed on soft PDMS\nsubstrates can be mechanically stabilized, maintaining a consistent percolation network even under\nrepeated tensile bending (up to 350 cycles at a bending radius of 20 mm; Supplementary Fig. 21b).\nIn Fig. 4, the notations SF020, SF002, and SF film appear in several graphs. These abbreviations\nare defined only in Supplementary Table 1. They should be clearly defined in the main text (e.g.,\nSF020 = 0.2 wt % SF, SF002 = 0.02 wt % SF) so readers need not consult the Supplementary\nInformation. Because dielectric thickness strongly affects capacitor performance, please also\nprovide approximate film thicknesses for these conditions.\nResponse:\nWe agree with the reviewer. We have defined the abbreviations directly in the main text and\nconsistently adopted them throughout the manuscript. We also agree that film thickness is\nimportant for interpreting capacitor performance. The ellipsometry-measured thicknesses used to\ncalculate capacitance density have now been added to the main text as detailed below.\nCorresponding revisions:\nPage 3, line 108 in the main manuscript\nPlease note that, unless otherwise specified, 0.02, 0.2, and 2.0 w/v% SF concentrations are denoted\nas SF002, SF020, and SF200, respectively, throughout the manuscript.\nPage 7, line 260 in the main manuscript\nThe average dielectric constants for pure crystallized SF films (thickness ~ 274 nm), optimal-SF\nadsorbed SiO2 NP capacitors (thickness ~ 321 nm), and pure SiO2 NP capacitors (thickness ~ 346\nnm) were 17.4 ± 0.9, 8.21 ± 0.5, and 5.97 ± 0.4 at 10 kHz, respectively, which are comparable to\nother solution-processed dielectrics33,34.\nThe Supplementary Information presents LSPR spectra of Au NPs and Au NRs on PDMS, but the\noptical structure of these plasmonic samples is not described—e.g., film thickness, particle density,\nor whether a monolayer or thicker layer was used. Since plasmonic response depends on particle\narrangement, a concise description of the coating morphology and thickness would make the\noptical data more meaningful.\nResponse:\nWe thank the reviewer for this helpful comment regarding the morphology of the plasmonic\nsamples.\nTo clarify, the synthesized Au NP (⌀ ~ 20 nm) dispersion was spin-coated directly onto\nPDMS substrates. Under these conditions, as shown in Supplementary Fig. 29b, the coated Au NP\nlayer is uniformly distributed with high surface density but without inter-particle coalescence.\nConsequently, the optical spectra exhibit LSPR peaks at ≈530 nm, consistent with typical values\nreported for isolated citrate-stabilized Au nanoparticles of similar size. We have revised the main\nmanuscript to describe these morphological features and added the SEM image to the\nSupplementary Information to provide a concise structural context for the LSPR data.\nCorresponding revisions:\nPage 8, line 303 in the main manuscript\nFor example, as shown in Supplementary Fig. 29, the Au NP and Au nanorod (NR) layers spin-\ncoated on PDMS form uniformly and closely packed monolayers, and their localized surface\nplasmon resonance (LSPR) spectra remain essentially unchanged despite SF adsorption.\nPage 35 in the Supplementary information\nSupplementary Figure 29. Negligible effect of SF adsorption on the localized surface plasmon\nresonance (LSPR).\na, The LSPR wavelengths of Au NPs with varying SF adsorption levels: no SF, 0.02 w/v%, 0.2\nw/v%, and 2.0 w/v%. b, SEM image showing top surface of Au NPs-coated PDMS. c, The LSPR\nwavelengths of Au NRs with varying SF adsorption levels.\nGeneral comment:\nThis manuscript describes the use of silk fibroin to assist with the formation of dense nanoparticle\nlayers. The authors show that silk fibroin can be non-specifically adsorbed onto the surface of\nnanoparticles, and that spincoating a nanoparticle/silk fibroin solution onto silicon yields a dense\nnanoparticle layer. The authors pinpoint a specific concentration range of silk fibroin that yields\nbest results in terms of conductivity and device performance (shown by a model nanoparticle-\nbased capacitor). The experiments are comprehensive, leveraging a variety of techniques for\nmolecular-scale characterization (e.g. of adhesion force, morphology, surface charge, device\nperformance metrics, etc). The data analysis is very competent, and I commend the authors for\npresenting a very clear manuscript.\nResponse:\nWe thank the reviewer for the positive overall assessment of our work and for the thoughtful,\nconstructive comments. In response, we have revised the manuscript to clarify the positioning and\nnovelty of our approach, refine our claims regarding applicability and stability, and improve the\ntransparency and reproducibility of the experimental conditions and analysis. We believe these\nchanges address all of the reviewer’s concerns and further strengthen the clarity and robustness of\nthe manuscript.\nOne of my major critique concerns the novelty and significance of the work. The adsorption of\nsilk fibroin to surfaces has been previously reported by Kaplan and colleagues as far back as 2005\n(https://doi.org/10.1021/la051862m, https://doi.org/10.1016/j.jconrel.2007.06.006,\nhttps://doi.org/10.1002/adfm.200901774 - this last one even reports coating nanoplatelets) and by\nZha and colleagues (https://pubs.rsc.org/en/content/articlepdf/2018/bm/c8bm01186a,\nhttps://doi.org/10.1021/acsbiomaterials.3c01042, https://doi.org/10.1021/acsami.4c07528). The\nprior literature shows many relevant aspects, such as the effect of pH, presence of ions,\nsecondary structure change upon adsorption, and adhesion (also measured by AFM force\nspectroscopy). However, there is insufficient discussion in the manuscript of the submitted work in\ncontext of these prior works.\nResponse:\nWe sincerely thank the reviewer for pointing out the extensive prior literature on the adsorption\nbehavior of SF. Indeed, a number of earlier works by Kaplan and colleagues (Langmuir 2005; J.\nControl. Release 2007; Adv. Funct. Mater. 2010) and by Zha and colleagues (Biomater. Sci. 2018;\nACS Biomater. Sci. Eng. 2023; ACS Appl. Mater. Interfaces 2024) have elegantly described the\nphysicochemical aspects of SF adsorption, including its dependence on pH, ionic strength, and\nsecondary structure transitions, and even its use in forming nanometric coatings via self-assembly.\nWe fully agree that these foundational studies have established the fundamental principles of silk–\nsurface interactions.\nOur study does not aim to revisit these well-established interfacial principles. Rather, it\nleverages them as a basis for a distinct objective: to translate SF adsorption into a process-enabling\nplatform for fully water-based, high-density nanoparticle (NP) nanofabrication on hydrophobic\nsubstrates, without thermal annealing or solvent-based activation. While previous studies\npredominantly examined SF adsorption in biological or materials-science contexts, they did not\ndemonstrate its use as a generalizable route to assemble electronically functional NP layers with\nsemiconductor-grade uniformity.\nBy exploiting SF as an amphiphilic adsorbate that mediates wetting, packing, and\ninterfacial adhesion, this work addresses a long-standing gap between aqueous-compatible\nbiomaterials and the precision required in nanoelectronic fabrication. This conceptual direction\nbuilds upon our recent report (Nat. Nanotechnol. 19, 1514–1520 (2024)), extending the role of SF\nfrom a molecular wetting agent to an adsorption-mediated NP assembly mechanism capable of\nproducing dense electronic and photonic interfaces entirely from water.\nstudies and to clarify how the present work advances them by providing the first demonstration of\ntheir translation into a high-precision, water-based nanoparticle self-assembly strategy for device-\nlevel biotic–abiotic integration.\nCorresponding revisions:\nPage 2, line 47 in the main manuscript\nExtensive foundational studies have established the physicochemical principles governing protein\nadsorption (e.g., silk fibroin (SF)) under aqueous conditions, including its effects on wettability,\nsecondary-structure transitions, and biological or material coatings13–18. While these works suggest\nthat protein adsorption may offer a natural route for forming biocompatible interfaces, they focus\nprimarily on fundamental interfacial behavior. Building upon this knowledge, recent efforts have\nbegun translating such phenomena into aqueous nanoprocessing strategies19,20. However, these\napproaches still rely on high-temperature annealing or solvent activation. A fully water-based route\ncapable of producing high-density, electronically functional NP layers without post-thermal\nprocessing remains unaddressed.\nThe ability of silk fibroin to adsorb to surfaces, and that absorption amount can be \"tuned\" by\nchanging silk fibroin concentration, is unsurprising, as this is typical macromolecular adsorption\nbehavior. I think the novelty of this manuscript is the identification of a silk fibroin concentration\nthat can give just enough \"stick\" to the nanoparticles to assist with forming a dense, conductive\nlayer. However, I would like a better discussion in the manuscript about how silk fibroin compares\nto other (potentially cheaper) molecules that can modify wettability, surface tension, and adhesion\n(such as surfactants, block copolymers, amphiphilic, non-amphiphilic polypeptides,\noligo/polysaccharides, etc). Is there something special about the silk fibroin sequence that allows\nto behave this way? How does the use of silk fibroin stack up against other methods of\nimproving nanoparticle wettability? This discuss is rather vague in the manuscript.\nResponse:\nWe thank the reviewer for raising this important point regarding how SF compares with other\nwettability-modifying or adhesion-modulating agents. As shown in our previous work (Nat.\nNanotechnol. 19, 1514–1520 (2024)), SF exhibits an interfacial behavior distinct from\nconventional surfactants, block copolymers, or polysaccharides: it spontaneously adsorbs onto\ndiverse solid surfaces without substantially altering bulk rheology or surface tension, enabling\nwetting improvement even at minute concentrations. This arises from the sequence-encoded\namphiphilicity of SF—alternating hydrophobic Gly–Ala–rich domains and hydrophilic, charged\nspacer regions—which allows simultaneous hydrophobic association, hydrogen bonding, and π-\ntype interactions with chemically heterogeneous substrates.\nIn the present study, we therefore focus not on re-benchmarking adsorption efficiency\nagainst synthetic surfactants, but on identifying the process window in which adsorbed SF provides\nsufficient interparticle cohesion to form dense, electrically reliable NP layers entirely from water.\nWhile commercial surfactants or polymeric stabilizers can improve NP dispersibility, their residual\ninsulating or ionic species are known to degrade charge transport unless removed by annealing or\npurification (J. Phys. Chem. C. 2013, 117, 11744–11749; Nat. Commun. 8, 1202 (2017); Chem.\nMater. 2021, 33, 4551–4557). In contrast, optimal SF adsorption yields uniform and conductive\nNP assemblies without post-processing, and its biocompatible, aqueous nature is inherently\naligned with sustainable and bio-integrated device fabrication.\nWe have revised the manuscript to clarify these distinctions and to better articulate the\nmolecular basis and practical advantages of SF-mediated nanofabrication.\nCorresponding revisions:\nThis study focuses on the adsorption-mediated NP-assembly behavior of SF within the optimal\nconcentration regime required for electronic functionality, rather than benchmarking against\nconventional surfactants whose effects on wettability and rheology have been addressed\npreviously19.\nThe claim that these devices can perform well in a biological setting is perhaps a bit of a stretch.\nSilk fibroin will swell (increasing the distance between nanoparticles) and be degraded over time\nif it is in contact with biological fluids. The electronic properties will certainly change over time.\nI don't see any mention of using an alcohol or annealing post-treatment of the films, which means\nthat the silk fibroin will likely dissolve in an aqueous environment.\nResponse:\nWe thank the reviewer for raising this important point regarding the stability of SF in aqueous and\nbiological environments. We fully agree that amorphous SF is prone to swelling and gradual\ndegradation in such settings, which would indeed lead to unstable electronic performance.\nConsistent with this concern, we observed that devices based on non-crystallized (amorphous) SF\nlayers exhibited significant humidity-induced drift.\nTo mitigate this, all SF-adsorbed NP assemblies used for electrical characterization in this\nwork were subjected to a brief methanol post-treatment (50 µL, 2000 rpm, 60 s) prior to testing,\nand it was described in the original main manuscript’s discussion section and method section (see\npage 7, line 248; page 11, line 442 in the revised main manuscript). This treatment induces β-\nsheet–rich crystallization of the SF adsorption layer, which is known to suppress swelling and\ndissolution, thereby stabilizing the interparticle packing and device characteristics under ambient\nhumidity, and allowing us to characterize the electrical performance reliably and reproducibly.\nI also have a few technical questions. Can the AFM IR data be deconvoluted to get information\nabout secondary structure composition? The signal at 1650 cm-1 is a random coil/alpha helix\nsignal, but I would expect there to be more beta-sheets in silk fibroin, particularly if this material\nis expected to not dissolve in aqueous environments (see https://doi.org/10.1021/acsami.4c07528\nfor similar AFM-IR analysis)\nResponse:\nWe appreciate the reviewer’s insightful comment regarding the secondary-structure composition\nof SF in the AFM-IR spectra. As shown in Supplementary Fig. 5, the AFM-IR measurements\nprovide characteristic IR fingerprints of SF films and SF-adsorbed PSNP coatings, and in principle\nthese spectra could be deconvoluted to estimate secondary-structure fractions, as demonstrated in\nprior work (e.g., ACS Appl. Mater. Interfaces 2024, 16, 43, 58121–58134).\nIn this study, however, the primary objective of the AFM-IR experiment was to spatially\nmap the presence and distribution of SF on NP surfaces, rather than to quantitatively resolve β-\nsheet content. Importantly, no post-crystallization (methanol) treatment was applied to the AFM-\nIR samples; we intentionally examined the as-adsorbed, amorphous SF layers to avoid altering\ntheir interfacial morphology prior to mapping. Under these conditions, a dominant amide I band\nnear 1650 cm-1, that is assigned to random-coil/α-helix conformations, is expected and consistent\nwith a largely amorphous SF adsorption layer.\nFor clarity, we have revised the Methods section to explicitly state that the SF films and\nSF-adsorbed PSNP assemblies used for AFM-IR characterization were not subjected to methanol-\ninduced crystallization.\nCorresponding revisions:\nPage 10, line 381 in the main manuscript\nBoth the SF films and the SF-adsorbed PSNPs assemblies were not subjected to post-\ncrystallization treatment.\nSome parameters critical to macromolecular adsorption are not included in the manuscript, such\nas molecular weight (and distribution) of the silk fibroin, solution pH (whether it was buffered or\nnot), and ion content. Without this information, reproducing the results (e.g. using a different silk\nfibroin source/batch, in a different lab, etc) will likely be very difficult.\nResponse:\nWe thank the reviewer for underscoring the importance of molecular-weight distribution, pH, and\nionic strength for macromolecular adsorption and reproducibility. In this study, we exclusively\nused 30-min boiled SF, whose molecular-weight distribution (~219–258 kDa) has been fully\ncharacterized in our prior work (Nat. Nanotechnology, 19, 1514-1520 (2024)). This specific SF\nbatch was chosen to maintain a consistent chain-length regime known to support robust interfacial-\nenergy modulation and adsorption, and all experiments in the present manuscript were performed\nusing this same material.\nUnless otherwise noted, all SF solutions and NP–SF mixtures were prepared in unbuffered\ndeionized water with no added salts, yielding near-neutral pH conditions (typically pH 6.8–7.2\nimmediately after mixing). This choice avoids ionic species that could interfere with NP-based\nelectronic measurements. For ζ-potential characterization only, 1× phosphate-buffered saline (PBS,\npH 7.4) was added to ensure reproducible electrokinetic measurements.\nWe have revised the Methods to explicitly include these parameters so that the adsorption\nand assembly conditions can be reliably reproduced across laboratories.\nCorresponding revisions:\nPage 9, line 342 in the main manuscript\nThe 30 min-boiled SF prepared in this manner exhibits a molecular weight distribution of ~219-\n258 kDa19.\nPage 9, line 349 in the main manuscript\nUnless otherwise noted, all suspensions were prepared in unbuffered DIW with no added salts/ions\nand used at room temperature under near-neutral pH (typically pH 6.8–7.2 immediately after\nmixing).\nPage 11, line 408 in the main manuscript\nTo maintain a pH, 100 μL of 1X phosphate-buffered saline (PBS, pH 7.4) was added to each tested\ndispersion.\nThe coating process is a bit confusing to me. Was non-adsorbed silk fibroin washed away (for\nexample, but spinning down the nanoparticles then resuspending in clean water) before spin\ncoating? There will obviously be excess silk fibroin between particles in the case of higher silk\nconcentration if there's no wash step. Incorporating a wash step may mean that the coating process\nis less sensitive to the concentration of the adsorption solution. Furthermore, if the non-adsorbed\nsilk fibroin is still present in the spin coating step, the surface tension may be different with\ndifferent concentrations, which may affect the outcomes of the spin coating.\nResponse:\nWe appreciate the reviewer’s question regarding the coating procedure. We intentionally did not\ninclude a wash step as described by the reviewer, because preserving the native adsorption\nequilibrium between SF and NPs is essential to reveal the concentration-dependent assembly\nbehavior observed in our water-based process. In this process, the adsorbed SF layer contributes\nto particle-to-particle adhesion, enhancing NP packing, while a minor residual free-SF fraction\nsubtly improves aqueous wetting on otherwise non-wettable surfaces such as PTFE and PDMS\n(Nat. Nanotechnol. 19, 1514–1520 (2024)). Together, these effects define an optimal window for\nNP assembly and coating. Below this window, insufficient SF adsorption and limited free-SF lead\nto poor wetting and sparse coverage, whereas above it, excess free SF accumulates in interstitial\nregions, increasing film thickness and interparticle spacing, which can degrade device performance\nand reproducibility (as shown in Fig. 3 and Fig. 4). This mechanism is also consistent with our\ninterfacial adhesion measurements, which demonstrate that even minute SF additions effectively\nregulate interfacial energy and enable aqueous wetting on hydrophobic substrates (Fig. 2).\nRegarding surface tension and viscosity, this study predominantly investigates ≤ 0.2 w/v%\nSF (the optimal/insufficient regime), with 2.0 w/v% included as a deliberately high-SF condition.\nIn the dilute regime, bulk rheological changes are negligible and both viscosity and surface tension\nremain comparable to those of the solvent, as reported previously (Macromol. Biosci. 8, 1006–\n1018, 2008). Despite the minimal bulk changes, interfacial adsorption governs the coating outcome\nthrough the mechanism described above. At 2.0 w/v%, a larger unbound SF fraction can both\naccumulate in interstitial regions and measurably alter solution rheology, jointly increasing film\nthickness—hence our focus on the optimal adsorption window in device-level comparisons (Fig.\n4).\nwas performed and that dispersions were used as prepared to preserve adsorption equilibrium. Also,\nwe also added a brief note in the main manuscript to better describe the coating mechanism.\nCorresponding revisions:\nPage 6, line 235 in the main manuscript\nIn this aqueous process, the coating behavior is jointly governed by an interfacially adsorbed SF\nlayer that defines NP assembly and by a minor residual free-SF fraction that modulates\n19,20\nwetting . This interplay establishes an optimal adsorption window in which film coverage and\nuniformity are maximized.\nPage 9, line 351 in the main manuscript\nNo wash/centrifugation step was applied to remove non-adsorbed free SF chains from the NP\nsuspensions.\nThe adhesion measurements in AFM were performed using a particle on the AFM tip and a flat\nhydrophobic substrate. Is this an acceptable model for the suspended nanoparticle system, where\nboth surfaces have substantial curvature? I think this model needs to be justified by at least showing\nthat silk fibroin coating thicknesses and secondary structures are similar on the flat surface vs the\ncurved nanoparticle surface.\nResponse:\nWe thank the reviewer for this important comment regarding the validity of using a curved–flat\nAFM geometry to represent the interactions between two SF-adsorbed nanoparticles (curved–\ncurved). We address this in two parts: (i) whether SF forms comparable adsorption layers on flat\nand curved hydrophobic surfaces, and (ii) whether the curved–flat geometry is appropriate for\nextracting concentration-dependent interaction trends.\n(i) Similarity of SF adsorption on flat vs. curved surfaces\nOur data show that SF adsorption layers form with comparable thicknesses and conformations on\nboth flat and curved surfaces. TEM analysis of SF020-adsorbed nanoparticles reveals an\nadsorption layer of ~10 nm (see Fig. 1d), while independent AFM force–distance measurements\non flat hydrophobic surfaces show a sharp onset of steric repulsion at ~10 nm (Supplementary Fig.\n10b), indicating that the SF layer formed on the flat substrate has nearly identical thickness.\nMoreover, the radius of gyration of SF in solution (<6 nm, Soft Matter, 6, 4389-4395 (2010)) is\norders of magnitude smaller than the radius of the AFM probe (~1 μm). Thus, at the molecular\nscale relevant to SF adsorption, the probe surface is effectively flat, and curvature does not alter\nSF chain conformation or secondary-structure formation. Together, these results directly support\nthe reviewer’s request to verify that the SF adsorption layer formed on flat surfaces is\nrepresentative of that on nanoparticles.\n(ii) Appropriateness of the curved–flat model\nAlthough the AFM geometry differs from the NP–NP configuration, it is fully appropriate for\nquantifying how silk fibroin modulates interfacial interactions. Under the Derjaguin\napproximation (Nature 353, 239-241 (1991)), the measured force between two bodies can be\nwritten as: F ≈ 2πR W\neff\nwhere R contains only the geometric contribution (curvature), while W is the interaction energy\neff\nper unit area determined by the SF adsorption layer. Because the adsorption thickness and\nsecondary structure of SF are comparable on flat and curved surfaces (as shown in part (i)), W is\n28\nexpected to be the same in both geometries. Thus, while absolute forces will differ due to different\nR , the concentration-dependent trends—our primary interest—directly reflect changes in W and\neff\nare validly captured using the curved–flat configuration.\nWe have added Supplementary Note3 to explicitly state this rationale.\nCorresponding revisions:\nPage 5 in the Supplementary information\nSupplementary Note 3. Validity of the curved–flat AFM geometry for interpreting SF-mediated\nnanoparticle interactions\nTo examine how SF adsorption modulates interparticle interactions in water-based nanoparticle\nassemblies, we employed a colloidal-probe AFM configuration in which a spherical particle on\nthe cantilever interacts with a flat hydrophobic substrate. Although this geometry differs from the\ncurved–curved configuration of two nanoparticles, the AFM model is appropriate because the\nstructural features of the SF adsorption layer are comparable across both surfaces.\nTEM imaging of SF020-adsorbed nanoparticles shows an adsorption layer with a thickness\nof approximately 10 nm (Fig. 1d). Independent AFM force–distance measurements on SF-coated\nflat hydrophobic substrates exhibit a steric-repulsion onset at a similar separation (Supplementary\nFig. 10b), indicating that the effective thickness of the SF adsorption layer is the same on both flat\nand curved interfaces. This agreement suggests that the adsorption process, including chain\nconformation and secondary-structure formation, is not strongly influenced by surface curvature.\nThis is consistent with the molecular dimensions of SF in aqueous environments: the radius of\ngyration of SF in solution (< 6 nm)5 is far smaller than the radius of the AFM colloidal probe (~1\nμm), making the probe surface effectively flat from the viewpoint of an adsorbing SF chain.\nThe measured AFM interaction force can be interpreted using the Derjaguin\napproximation6, in which the force between two bodies is expressed as follows:\n𝐹 ≈ 2𝜋𝑅 𝑊 (3)\neff\nwhere R represents the geometric contribution and W denotes the interaction energy per unit area\neff\ngoverned by the SF adsorption layer. Because the structural characteristics of the adsorption layer\nare comparable across the two geometries, the interaction energy W is expected to be equivalent.\nWhile the absolute force magnitude depends on geometry through R , the concentration-\neff\ndependent trends, which are our primary interest, reflect changes in W, and therefore the curved–\nflat AFM measurements reliably capture the interfacial behavior relevant to nanoparticle–\nnanoparticle contact.\nThese considerations support the validity of the AFM measurement scheme for analyzing SF-\nmediated interaction trends in suspended nanoparticle systems, which is described in Fig 2 and\nSupplementary Fig. 10-12.\nI appreciate the authors’ thorough, clear, and scientifically rigorous responses to all\nof my comments. Their revisions—both in the main manuscript and the\nsupplementary materials—demonstrate a sincere effort to address each point with\ncare, and the additional analyses and clarifications have substantially strengthened\nthe work. I am fully satisfied with the authors’ responses, and in my view, the\nmanuscript now meets the standards for publication in Nature Communications. I\nrecommend acceptance in its current form.\nWe thank the reviewer for the thoughtful review on our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed my previous concerns in this current revision.\nWe appreciate the reviewer for the thorough investigation on our manuscript.\n1"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I appreciate the authors’ thorough, clear, and scientifically rigorous responses to all of my comments. Their revisions—both in the main manuscript and the supplementary materials—demonstrate a sincere effort to address each point with care, and the additional analyses and clarifications have substantially strengthened the work. I am fully satisfied with the authors’ responses, and in my view, the manuscript now meets the standards for publication in Nature Communications. I recommend acceptance in its current form.", "reviewer_3": "The authors have addressed my previous concerns in this current revision."}, "author_response": ""}]

Decision letter