Molecular organization of fibroin heavy chain and mechanism of fibre formation in Bombyx mori.

Fibroins' transition from liquid to solid is fundamental to spinning and underpins the impressive native properties of silk. Herein, we establish a fibroin heavy chain fold for the Silk-I polymorph, which could be relevant for other similar proteins, and explains mechanistically the liquid-to-solid transition of this silk, driven by pH reduction and flow stress. Combining spectroscopy and modelling we propose that the liquid Silk-I fibroin heavy chain (FibH) from the silkworm, Bombyx mori, adopts a newly reported ß-solenoid structure. Similarly, using rheology we propose that FibH N-terminal domain (NTD) templates reversible higher-order oligomerization driven by pH reduction. Our integrated approach bridges the gap in understanding FibH structure and provides insight into the spatial and temporal hierarchical self-assembly across length scales. Our findings elucidate the complex rheological behaviour of Silk-I, solutions and gels, and the observed liquid crystalline textures within the silk gland. We also find that the NTD undergoes hydrolysis during standard regeneration, explaining key differences between native and regenerated silk feedstocks. In general, in this study we emphasize the unique characteristics of native and native-like silks, offering a fresh perspective on our fundamental understanding of silk-fibre production and applications.

Electrical stimulation enhances sciatic nerve regeneration using a silk-based conductive scaffold beyond traditional nerve guide conduits.

Despite recent advancements in peripheral nerve regeneration, the creation of nerve conduits with chemical and physical cues to enhance glial cell function and support axonal growth remains challenging. This study aimed to assess the impact of electrical stimulation (ES) using a conductive nerve conduit on sciatic nerve regeneration in a rat model with transection injury. The study involved the fabrication of conductive nerve conduits using silk fibroin and Au nanoparticles (AuNPs). Collagen hydrogel loaded with green fluorescent protein (GFP)-positive adipose-derived mesenchymal stem cells (ADSCs) served as the filling for the conduit. Both conductive and non-conductive conduits were applied with and without ES in rat models. Locomotor recovery was assessed using walking track analysis. Histological evaluations were performed using H&E, luxol fast blue staining and immunohistochemistry. Moreover, TEM analysis was conducted to distinguish various ultrastructural aspects of sciatic tissue. In the ES + conductive conduit group, higher S100 (p < 0.0001) and neurofilament (p < 0.001) expression was seen after 6 weeks. Ultrastructural evaluations showed that conductive scaffolds with ES minimized Wallerian degeneration. Furthermore, the conductive conduit with ES group demonstrated significantly increased myelin sheet thickness and decreased G. ratio compared to the autograft. Immunofluorescent images confirmed the presence of GFP-positive ADSCs by the 6th week. Locomotor recovery assessments revealed improved function in the conductive conduit with ES group compared to the control group and groups without ES. These results show that a Silk/AuNPs conduit filled with ADSC-seeded collagen hydrogel can function as a nerve conduit, aiding in the restoration of substantial gaps in the sciatic nerve with ES. Histological and locomotor evaluations indicated that ES had a greater impact on functional recovery compared to using a conductive conduit alone, although the use of conductive conduits did enhance the effects of ES.

Studies on the potential risk of amyloidosis from exposure to cultured fibril from silk fibroin.

Amyloid A (AA) amyloidosis is induced by administering amyloid fibrils to animals under inflammatory conditions. Silk fibroin (SF), the main component of silk threads, forms amyloid-like fibrils and has been previously reported to induce AA amyloidosis in mice. In this study, SF was cultured in ethanol solution, and after confirming fibril formation through thioflavin T assay, Congo red assay, and observation under electron microscopy, cultured SF ethanol solutions were administered to mice via various routes to investigate the induction of target organs and amyloidosis. As a result, cultured SF ethanol solutions were confirmed to reach the lungs and spleen, but no amyloid deposition was observed. While SF forms amyloid-like fibril structures through cultivation in ethanol solution, its amyloid-enhancing factor (AEF) activity is considered low in mice.

Overview and Evolution of Insect Fibroin Heavy Chain (FibH).

The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39-45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

Silk-gelatin hybrid hydrogel: a potential carrier for RNA therapeutics.

RNA-based therapeutics have exhibited remarkable potential in targeting genetic factors for disease intervention, exemplified by recent mRNA vaccines for COVID-19. Nevertheless, the intrinsic instability of RNA and challenges related to its translational efficiency remain significant obstacles to the development of RNA as therapeutics. This study introduces an innovative RNA delivery approach using a silk fibroin (SF) and positively charged gelatin (Gel) hydrogel matrix to enhance RNA stability for controlled release. As a proof of concept, whole-cell RNA was incorporated into the hydrogel to enhance interactions with RNA molecules. Additionally, molecular modeling studies were conducted to explore the interactions between SF, collagen, chitosan (Chi), and the various RNA species including ribosomal RNAs (28S, 18S, 8.5S, and 5S rRNAs), transfer RNAs (tRNA-ALA, tRNA-GLN, and tRNA-Leu), as well as messenger RNAs (mRNA-GAPDH, mRNA-ß actin, and mRNA-Nanog), shedding light on the RNA-polymer interaction and RNA stability; SF exhibits a more robust interaction with RNA compared to collagen/gel and chitosan. We confirmed the molecular interactions of SF and RNA by FTIR and Raman spectroscopy, which were further supported by AFM and contact angle measurement. This research introduces a novel RNA delivery platform and insights into biopolymer-RNA interactions, paving the way for tailored RNA delivery systems in therapeutics and biomedical applications.

Tailoring silk fibroin hydrophilicity and physicochemical properties using sugar alcohols for medical device coatings.

This study explores the modification of silk fibroin films for hydrophilic coating applications using various sugar alcohols. Films, prepared via solvent casting, incorporated glycerol, sorbitol, and maltitol, revealing distinctive transparency and UV absorption characteristics based on sugar alcohol chemical structures. X-ray diffraction confirmed a silk I to silk II transition influenced by sugar alcohols. Glycerol proved most effective in enhancing the ß-sheet structure. The study also elucidated a conformational shift towards a ß-sheet structure induced by sugar alcohols. Silk fibroin-sugar alcohol blind docking and sugar alcohol-sugar alcohol blind docking investigations were conducted utilizing the HDOCK Server. The computer simulation unveiled the significance of size and hydrogen bonding characteristics inherent in sugar alcohols, emphasizing their pivotal role in influencing interactions within silk fibroin matrices. Hydrophilicity of ozonized silicone surfaces improved through successful coating with silk fibroin films, particularly glycerol-containing ones, resulting in reduced contact angles. Strong adhesion between silk fibroin films and ozonized silicone surfaces was evident, indicating robust hydrogen bonding interactions. This comprehensive research provides crucial insights into sugar alcohols' potential to modify silk fibroin film crystalline structures, offering valuable guidance for optimizing their design and functionality, especially in silicone coating applications.

Time-programmed release of curcumin and Zn2+ from multi-layered RSF coating modified PET graft for improvement of graft-host integration.

Artificial graft serves as the primary grafts used in the clinical management of sports-related injuries. Until now, optimizing its graft-host integration remains a great challenge due to the excessive inflammatory response during the inflammatory phase, coupled with an absence of tissue-inductive capacity during the regeneration phase. Here, a multi-layered regenerated silk fibroin (RSF) coating loaded with curcumin (Cur) and Zn2+ on the surface of the PET grafts (Cur@Zn2+@PET) was designed and fabricated for providing time-matched regulation specifically tailored to address issues arising at both inflammatory and regeneration phases, respectively. The release of Cur and Zn2+ from the Cur@Zn2+@PET followed a time-programmed pattern in vitro. Specifically, cellular assays revealed that Cur@Zn2+@PET initially released Cur during the inflammatory phase, thereby markedly inhibit the expression of inflammatory cytokines TNF-a and IL-1ß. Meanwhile, a significant release of Zn2+ was major part during the regeneration phase, serving to induce the osteogenic differentiation of rBMSC. Furthermore, rat model of anterior cruciate ligament reconstruction (ACLR) showed that through time-programmed drug release, Cur@Zn2+@PET could suppress the formation of fibrous interface (FI) caused by inflammatory response, combined with significant new bone (NB) formation during regeneration phase. Consequently, the implementation of the Cur@Zn2+@PET characterized by its time-programmed release patterns hold considerable promise for improving graft-host integration for sports-related injuries.

A hybrid structure based on silk fibroin/PVA nanofibers and alginate/gum tragacanth hydrogel embedded with cardamom extract.

Hybrid structures made of natural-synthetic polymers have been interested due to high biological features combining promising physical-mechanical properties. In this research, a hybrid dressing consisting of a silk fibroin (SF)/polyvinyl alcohol (PVA) nanofibers and sodium alginate (SA)/gum tragacanth (GT) hydrogel incorporating cardamom extract as an antibacterial agent was prepared. Accordingly, SF was extracted from cocoons followed by electrospinning in blend form with PVA (SF/PVA ratio: 1:1) under the voltage of 18 kV and the distances of 15 cm. The SEM images confirmed the formation of uniform, bead free fibers with the average diameter of 199 ± 28 nm. FTIR and XRD results revealed the successful extraction of SF and preparation of mixed fibrous mats. Next, cardamom oil extract-loaded SA/GT hydrogel was prepared and the nanofibrous structure was placed on the surface of hydrogel. SEM analysis depicted the uniform morphology of hybrid structure with desirable matching between two layers. TGA analysis showed desired thermal stability. The swelling ratio was found to be 1251% after 24 h for the hybrid structure and the drug was released without any initial burst. MTT assay and cell attachment results showed favorable biocompatibility and cell proliferation on samples containing extract, and antibacterial activity values of 85.35% against S. aureus and 75% against E. coli were obtained as well. The results showed that the engineered hybrid nanofibrous-hydrogel film structure incorporating cardamom oil extract could be a promising candidate for wound healing applications and skin tissue engineering.

Mild preparation of hyaluronic acid/silk fibroin sponges by modified crosslinking method.

The composites composed of hyaluronic acid (HA) and silk fibroin (SF) exhibit great potential in diverse biomedical applications. However, the utilization of commercial crosslinkers such as 1,4-butanediol diglycidyl ether (BDDE) for crosslinking HA typically necessitates harsh conditions involving strong alkaline, which greatly limits its potential applications. In this study, a mild modified approach was developed to fabricate HA/SF blend sponges crosslinked by BDDE without alkaline conditions. The blend solutions were cryo-concentrated to induce crosslinking reactions. The mechanism of freezing crosslinking was elucidated by investigating the effects of ice crystal growth and HA molecular weight on the degree of crosslinking. The results revealed that HA achieved efficient crosslinking when its molecular weight exceeds 1000 kDa and freezing temperatures ranged from -40 °C to -20 °C. After introducing SF, multiple crosslinks were formed between SF and HA chains, producing water-stable porous sponges. The SEM results demonstrated that the introduction of SF effectively enhanced the interconnectivity between macropores through creating subordinate holes onto the pores wall. Raising the SF content significantly enhanced compression strength, resistance to enzymatic degradation and cell viability of blend sponges. This study provides a novel strategy for designing bioactive HA/SF blend sponges as substitutes for tissue repair and wound dressing.

Dry and wet wrinkling of a silk fibroin biopolymer by a shape-memory material with insight into mechanical effects on secondary structures in the silk network.

Surface wrinkling provides an approach to modify the surfaces of biomedical devices to better mimic features of the extracellular matrix and guide cell attachment, proliferation, and differentiation. Biopolymer wrinkling on active materials holds promise but is poorly explored. Here we report a mechanically actuated assembly process to generate uniaxial micro-and nanosized silk fibroin (SF) wrinkles on a thermo-responsive shape-memory polymer (SMP) substrate, with wrinkling demonstrated under both dry and hydrated (cell compatible) conditions. By systematically investigating the influence of SMP programmed strain magnitude, film thickness, and aqueous media on wrinkle stability and morphology, we reveal how to control the wrinkle sizes on the micron and sub-micron length scale. Furthermore, as a parameter fundamental to SMPs, we demonstrate that the temperature during the recovery process can also affect the wrinkle characteristics and the secondary structures in the silk network. We find that with increasing SMP programmed strain magnitude, silk wrinkled topographies with increasing wavelengths and amplitudes are achieved. Furthermore, silk wrinkling is found to increase ß-sheet content, with spectroscopic analysis suggesting that the effect may be due primarily to tensile (e.g., Poisson effect and high-curvature wrinkle) loading modes in the SF, despite the compressive bulk deformation (uniaxial contraction) used to produce wrinkles. Silk wrinkles fabricated from sufficiently thick films (roughly 250 nm) persist after 24 h in cell culture medium. Using a fibroblast cell line, analysis of cellular response to the wrinkled topographies reveals high viability and attachment. These findings demonstrate use of wrinkled SF films under physiologically relevant conditions and suggest the potential for biopolymer wrinkles on biomaterials surfaces to find application in cell mechanobiology, wound healing, and tissue engineering.

Effect of Phosphate on the Molecular Properties, Interactions, and Assembly of Engineered Spider Silk Proteins.

Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to ß-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced ß-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of ß-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.

Biodegradable quaternized silk fibroin sponge with highly uniform pore structure for traumatic hemostasis and anti-infection.

Developing a biodegradable sponge with rapid shape recovery and potent antibacterial and coagulation properties for traumatic hemostasis and anti-infection remains challenging. Herein, we fabricated quaternized silk fibroin (SF) sponges by freeze-drying under a constant cooling rate and modification with quaternary ammonium groups. We found the constant cooling rate enabled the sponges with a highly uniform pore structure, which provided excellent self-elasticity and shape recovery. Decoration with quaternary ammonium groups enhanced blood cells adhesion, aggregation, and activation, as well as resistance to infections from Staphylococcus aureus and Escherichia coli. The SF sponge had superior hemostatic capacity to gauze and commercial gelatin sponge in different hemorrhage models. The SF sponge exhibited favorable biodegradability and biocompatibility. Moreover, The SF sponge also promoted host cell infiltration, capillary formation, and tissue ingrowth, suggesting its potential for guiding tissue regeneration. The developed SF sponge holds great application prospects for traumatic hemostasis, anti-infection, and guiding tissue regeneration.

Heat-Insulated Regenerated Fibers with UV Resistance: Silk Fibroin/Al2O3 Nanoparticles.

The various wastes generated by silkworm silk textiles that are no longer in use are increasing, which is causing considerable waste and contamination. This issue has attracted widespread attention in countries that use a lot of silk. Therefore, enhancing the mechanical properties of regenerated silk fibroin (RSF) and enriching the function of silk are important directions to expand the comprehensive utilization of silk products. In this paper, the preparation of RSF/Al2O3 nanoparticles (NPs) hybrid fiber with different Al2O3 NPs contents by wet spinning and its novel performance are reported. It was found that the RSF/Al2O3 NPs hybrid fiber was a multifunctional fiber material with thermal insulation and UV resistance. Natural light tests showed that the temperature rise rate of RSF/Al2O3 NPs hybrid fibers was slower than that of RSF fibers, and the average temperature rose from 29.1 °C to about 35.4 °C in 15 min, while RSF fibers could rise to about 40.1 °C. UV absorption tests showed that the hybrid fiber was resistant to UV radiation. Furthermore, the addition of Al2O3 NPs may improve the mechanical properties of the hybrid fibers. This was because the blending of Al2O3 NPs promoted the self-assembly of ß-sheets in the RSF reaction mixture in a dose-dependent manner, which was manifested as the RSF/Al2O3 NPs hybrid fibers had more ß-sheets, crystallinity, and a smaller crystal size. In addition, RSF/Al2O3 NPs hybrid fibers had good biocompatibility and durability in micro-alkaline sweat environments. The above performance makes the RSF/Al2O3 NPs hybrid fibers promising candidates for application in heat-insulating and UV-resistant fabrics as well as military clothing.

The investigation of the interaction between fluorescent carbon dots labeling silk fibroin using a fluorescence microscope-surface plasmon resonance system.

The use of fluorescent carbon dots (CDs) as highly precise biolabeling probes has been widespread in the fields of live cell imaging and protein labeling due to their small size and excellent photoluminescence ability to accurately target specific molecules with surface chemical properties. However, there was a lack of research on the interaction between CDs and labeled molecules. In this work, we presented a novel investigation strategy, the fluorescence microscopy-surface plasmon resonance (FM-SPR) system, which combined the use of fluorescence microscopy and wavelength modulation surface plasmon resonance to study the interaction between CDs and labeled molecules in real-time. Using this system, simultaneously recorded the SPR signals and the fluorescence images on the surface of the FM-SPR sensor chip. We observed the dynamic curve and fluorescence images of the interaction between green emissive nitrogen-doped carbon dots (N-CDs) and silk fibroin (SF) in real-time. The kinetic parameters, the quantitative analysis, and the investigation of the binding could be achieved. The results showed a strong linear relationship between the change in SPR signals and the concentration of N-CDs, with a linear coefficient of 0.99913. The linear detection range was 2.5 µg/mL-100 µg/mL, and the real lowest detection limit reached 0.5 µg/mL. Additionally, the green fluorescence points in the imaging region on the FM-SPR sensor chip increased with the concentration of N-CDs, which was consistent with the change in SPR signals. Using this system we also acquired the association rate and dissociation rate of N-CDs to SF which were 2.65 × 10-5/s and 1.52 × 10-5/s, respectively. This demonstrated the effectiveness of our method in quantitatively analyzing SF labeled with N-CDs.

An engineered Pichia pastoris platform for the biosynthesis of silk-based nanomaterials with therapeutic potential.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyl-tRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

Mechanically robust and personalized silk fibroin-magnesium composite scaffolds with water-responsive shape-memory for irregular bone regeneration.

The regeneration of critical-size bone defects, especially those with irregular shapes, remains a clinical challenge. Various biomaterials have been developed to enhance bone regeneration, but the limitations on the shape-adaptive capacity, the complexity of clinical operation, and the unsatisfied osteogenic bioactivity have greatly restricted their clinical application. In this work, we construct a mechanically robust, tailorable and water-responsive shape-memory silk fibroin/magnesium (SF/MgO) composite scaffold, which is able to quickly match irregular defects by simple trimming, thus leading to good interface integration. We demonstrate that the SF/MgO scaffold exhibits excellent mechanical stability and structure retention during the degradative process with the potential for supporting ability in defective areas. This scaffold further promotes the proliferation, adhesion and migration of osteoblasts and the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. With suitable MgO content, the scaffold exhibits good histocompatibility, low foreign-body reactions (FBRs), significant ectopic mineralisation and angiogenesis. Skull defect experiments on male rats demonstrate that the cell-free SF/MgO scaffold markedly enhances bone regeneration of cranial defects. Taken together, the mechanically robust, personalised and bioactive scaffold with water-responsive shape-memory may be a promising biomaterial for clinical-size and irregular bone defect regeneration.

Silk fibroin sponge impregnated with fish bone collagen: A promising wound healing scaffold and skin tissue regeneration.

In the present study, porous silk fibroin sponges (SFS) were prepared using silk fibroin (SF), fish bone collagen (FBC), and olive oil (OO). The study investigates the potential use of using this sponge as skin tissue regeneration. The sponge was characterized for its physicochemical, mechanical, antimicrobial, and drug release properties. An in vitro study was carried out using human keratinocyte cell line (HaCaT). Biodegradation study using enzymatic method was carried out. The results showed that the mechanical properties such as tensile strength (23.40 ± 0.05 MPa), elongation at break (14.25 ± 0.02%), and water absorption (30.23 ± 0.01%) of the SFS were excellent, indicating promising performance. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays proved the biocompatible nature of the SFS. The SFS exhibited outstanding antibacterial properties against E. coli (4.72 ± 0.05 mm) and S. aureus (4.98 ± 0.07 mm). The developed SFS promote a promising solution for skin tissue regeneration and wound dressing.

Aptamer-free upconversion nanoparticle/silk biosensor system for low-cost and highly sensitive detection of antibiotic residues.

The detection of antibiotics is crucial for safeguarding the environment, ensuring food safety, and promoting human health. However, developing a rapid, convenient, low-cost, and sensitive method for antibiotic detection presents significant challenges. Herein, an aptamer-free biosensor was successfully constructed using upconversion nanoparticles (UCNPs) coated with silk fibroin (SF), based on Förster resonance energy transfer (FRET) and the charge-transfer effect, for detecting roxithromycin (RXM). A synergistic FRET efficiency was achieved by utilizing alizarin red and RXM complexes as energy acceptors, with UCNP as the energy donor, and immobilizing an ultrathin SF protein corona within 10 nm. The biosensor detects RXM in deionized water with high sensitivity primarily through monolayer adsorption, with a detection range of 1.0 nM-141.6 nM and a detection limit as low as 0.68 nM. The performance of this biosensor was compared with the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for detecting antibiotics in river water separately and a strong correlation between the two methods was observed. The biosensor exhibited long-term stability in aqueous solutions (up to 60 d) with no attenuation of fluorescence intensity. Furthermore, the biosensor's applicability extended to the highly sensitive detection of other antibiotics, such as azithromycin. This study introduces a low-cost, eco-friendly, and highly sensitive method for antibiotic detection, with broad potential for future applications in environmental, healthcare, and food-related fields.

Interfacial interactions between spider silk protein and cellulose studied by molecular dynamics simulation.

CONTEXT: Due to their excellent biocompatibility and degradability, cellulose/spider silk protein composites hold a significant value in biomedical applications such as tissue engineering, drug delivery, and medical dressings. The interfacial interactions between cellulose and spider silk protein affect the properties of the composite. Therefore, it is important to understand the interfacial interactions between spider silk protein and cellulose to guide the design and optimization of composites. The study of the adsorption of protein on specific surfaces of cellulose crystal can be very complex using experimental methods. Molecular dynamics simulations allow the exploration of various physical and chemical changes at the atomic level of the material and enable an atomic description of the interactions between cellulose crystal planes and spider silk protein. In this study, molecular dynamics simulations were employed to investigate the interfacial interactions between spider silk protein (NTD) and cellulose surfaces. Findings of RMSD, RMSF, and secondary structure showed that the structure of NTD proteins remained unchanged during the adsorption process. Cellulose contact numbers and hydrogen bonding trends on different crystalline surfaces suggest that van der Waals forces and hydrogen bonding interactions drive the binding of proteins to cellulose. These findings reveal the interaction between cellulose and protein at the molecular level and provide theoretical guidance for the design and synthesis of cellulose/spider silk protein composites. METHODS: MD simulations were all performed using the GROMACS-5.1 software package and run with CHARMM36 carbohydrate force field. Molecular dynamics simulations were performed for 500 ns for the simulated system.