Rational Design of Spider Silk Materials Genetically Fused with an Enzyme

Enzyme immobilization is an attractive route for achieving catalytically functional surfaces suitable for both continuous and repeated use. Herein, genetic engineering is used to combine the catalytic ability of a xylanase with the self‐assembly properties of recombinant spider silk, realizing silk materials with enzymatic activity. Under near‐physiological conditions, soluble xylanase‐silk fusion proteins assembled into fibers displaying catalytic activity. Also, a xylanase‐silk protein variant with the silk part miniaturized to contain only the C‐terminal domain of the silk protein formed fibers with catalytic activity. The repertoire of xylanase‐silk formats is further extended to include 2D surface coatings and 3D foams, also being catalytically active, showing the versatile range of possible silk materials. The stability of the xylanase‐silk materials is explored, demonstrating the possibility of storage, reuse, and cleaning with ethanol. Interestingly, fibers can also be stored dried with substantial residual activity after rehydration. Moreover, a continuous enzymatic reaction using xylanase‐silk is demonstrated, making enzymatic batch reactions not the sole possible implementation. The proof‐of‐concept for recombinantly produced enzyme‐silk, herein shown with a xylanase, implies that also other enzymes can be used in similar setups. It is envisioned that the concept of enzyme‐silk can find its applicability in, for example, multienzyme reaction systems or biosensors.     

Recombinant Spider Silk Forms Tough and Elastic Nanomembranes that are Protein‐Permeable and Support Cell Attachment and Growth

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers.     

Designed Multifunctional Spider Silk Enabled by Genetically Encoded Click Chemistry

Spider silk is recognized for its exceptional mechanical properties and biocompatibility, making it a versatile platform for developing functional materials. In this study, a modular functionalization strategy for recombinant spider silk is presented using SpyTag/SpyCatcher chemistry, a prototype of genetically encoded click chemistry. The approach involves AlphaFold2-aided design of SpyTagged spider silk coupled with bacterial expression and biomimetic spinning, enabling the decoration of silk with various SpyCatcher-fusion motifs, such as fluorescent proteins, enzymes, and cell-binding ligands. The silk threads can be coated with a silica layer using silicatein, an enzyme for silicification, resulting in a hybrid inorganic–organic 1D material. The threads installed with RGD or laminin cell-binding ligands lead to enhanced endothelial cell attachment and proliferation. These findings demonstrate a straightforward yet powerful approach to 1D protein materials.     

Meso‐Functionalization of Silk Fibroin by Upconversion Fluorescence and Near Infrared In Vivo Biosensing

In biomedical applications, it is very desirable to monitor the in vivo state of implanted devices, i.e., tracking the location, the state, and the interaction between the implanted devices and cell tissues. To achieve this goal, a generic strategy of soft materials meso‐functionalization is presented. This is to acquire silk fibroin (SF) materials with added functions, i.e., in vivo bioimaging/sensing. The functionalization is by 3D materials assembly of functional components, lanthanide(Ln)‐doped upconversion nanoparticles (UCNPs) on the mesoscopic scale to acquire upconversion fluorescent emission. To implement the meso‐functionalization, the surfaces of UCNPs are modified by the hydroxyl groups (? OH) from SiO₂ or polyethylene glycol coating layers, which can interact with the carbonyl groups (C?O) in SF scaffolds. The functionalized silk scaffolds are further implanted subcutaneously into mice, which allows the silk scaffolds to have fluorescent in vivo bioimaging and other biomedical functions. This material functionalization strategy may lead to the rational design of biomaterials in a more generic way.     

An Artificial Motion and Tactile Receptor Constructed by Hyperelastic Double Physically Cross-Linked Silk Fibroin Ionoelastomer

Ionotronic artificial motion and tactile receptor (i-AMTR) is essential to realize an interactive human-machine interface. However, an i-AMTR that effectively mimics the composition, structure, mechanics, and multi-functionality of human skin, called humanoid i-AMTR, is yet to be developed. To bridge this technological gap, this study proposes a strategy that combines molecular structure design and function integration to construct a humanoid i-AMTR. Herein, a silk fibroin ionoelastomer (SFIE) with double cross-linked molecular structure is designed to mimic the composition and structure of human skin, thereby resolving the conflict of stretchability, softness, and resilience, suffered by many previously reported ionotronics. Functionally, electromechanical sensing and triboelectricity-based tactile perception are integrated into SFIE, to enable simultaneous perception of both motion and tactile inputs. By further leveraging the machine learning and Internet of Things (IoT) techniques, the proposed SFIE-based humanoid i-AMTR precisely senses the movement of human body and accurately sortball objects made of different materials. Notably, the success rate for 610 sorting tests reaches as high as 92.3%. These promising results essentially demonstrate a massive potential of humanoid i-AMTR in the fields of sorting robots, rehabilitation medicine, and augmented reality.     

不同波长光照射血液诱发的荧光光谱研究

为研究波长因素在激光与血液相互作用中的影响 ,从健康人静脉采集血液 ,分别用 5 0 2nm ,5 30nm和 6 32 8nm波长光照射血液样品 ,同时用光栅光谱仪检测其荧光光谱。结果表明 ,5 30nm波长光在血液中引发的荧光辐射最强 ;5 0 2nm波长光在血液中引发的荧光辐射比较弱 ;6 32 8nm波长光照射血液 ,所产生的发射光谱在原激发光的长波和短波两个方向都有分布。这一结果提示 ,上述三种波长光与血液相互作用的过程有所不同 ,因而其对血液的生物效应也会有所差异。     

Reversible Fluorescent On–Off Recording in a Highly Transparent Polymeric Material Utilizing Fluorescent Resonance Energy Transfer (FRET) Induced by Heat Treatment

One productive technique for ultrahigh resolution readout of tiny regions is the measurement of the fluorescence signal of materials. A transparent polymeric materials whose fluorescence quantum yield is changed and recorded by thermally controlling the aggregation of fluoran dyes and developers with long alkyl chains has been developed. The recording medium can be fabricated easily by casting or coating recording materials. Fluorescence is observed after annealing at 363 K for about twelve seconds and then cooling to room temperature (RT), and quenched by annealing at 423 K for a few seconds and then quenching to RT. Nondestructive readout by excitation light with a fluorescent contrast of above 10 is achieved using red, green, and blue fluorescent dyes. Fluorescence on–off switching is induced by fluorescent resonance energy transfer (FRET) from a fluorescent dye to a colored fluoran dye in the recording material. Fluorescence was uniformly quenched in the visible region after erasing. Since the recording materials allow the penetration of laser light due to the presence of crystals smaller than the wavelength range of visible light in both the emission and quenching states, nondestructive readout of the fluorescent signal by two‐photon absorption is accomplished. This work provides an important stepping‐stone for achieving rewritable‐type near‐field optical storage or multilayer recording.     

Surface Features of Recombinant Spider Silk Protein eADF4(κ16)‐Made Materials are Well‐Suited for Cardiac Tissue Engineering

Cardiovascular diseases causing high morbidity and mortality represent a major socioeconomic burden. The primary cause of impaired heart function is often the loss of cardiomyocytes. Thus, novel therapies aim at restoring the lost myocardial tissue. One promising approach is cardiac tissue engineering. Previously, it is shown that Antheraea mylitta silk protein fibroin is a suitable material for cardiac tissue engineering, however, its quality is difficult to control. To overcome this limitation, the interaction of primary rat heart cells with engineered Araneus diadematus fibroin 4 (κ16) (eADF4(κ16)) is investigated here, which is engineered based on the sequence of ADF4 by replacing the glutamic acid residue in the repetitive unit of its core domain with lysine. The data demonstrate that cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells attach well to eADF4(κ16) films on glass coverslips which provide an engineered surface with a polycationic character. Moreover, eADF4(κ16) films have, in contrast to fibronectin films, no hypertrophic effect but allow the induction of cardiomyocyte hypertrophy. Finally, cardiomyocytes grown on eADF4(κ16) films respond to pro‐proliferative factors and exhibit proper cell‐to‐cell communication and electric coupling. Collectively, these data demonstrate that designed recombinant eADF4(κ16)‐based materials are promising materials for cardiac tissue engineering.     

Biomimetic Tendrils by Four Dimensional Printing Bimorph Springs with Torsion and Contraction Properties Based on Bio-Compatible Graphene/Silk Fibroin and Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)

Taking inspiration from plant tendril geometry, in this study, 4D bimorph coiled structures with an internal core of graphene nanoplatelets-modified regenerated silk and an external shell of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) are fabricated by 4D printing. Finite element simulations and experimental tests demonstrate that integrating these biomaterials with different coefficients of thermal expansion results in the temperature induced self-compression and torsion of the structure. The bimorph spring also exhibits reversible contractive actuation after exposure to water environment that paves its exploitation in regenerative medicine, since core materials also have been proven to be biocompatible. Finally, the authors validate their findings with experimental measurements using such springs for temperature-mediated lengthening of an artificial intestine.