Developing artificial spider silk with similar properties to primary silk has been a challenging task in materials science. In this study, we used a microfluidic device to create continuous fibers based on recombinant MaSp2 spiroprotein. This strategy combines ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induced β sheet formation. We found that a threshold shear stress of approximately 72 Pa is required for fiber formation, and the formation of β sheets depends on the presence of polyalanine blocks in the repeating sequence. The β content (29.2%) of the resulting MaSp2 fibers is comparable to that of a natural dragline with a shear stress requirement of 111 Pa, and interestingly, the polyalanine block has a limited effect on liquid-liquid phase separation and layered structure. The results of this study provide a new development idea for the shear-induced crystallization and layer-weaving relationship of spider silk, which is of great significance for the rational design of artificially spun fibers.
Spider Pull Shovel Silk is a protein-based biopolymer that embodies an unmatched combination of strength and flexibility and has attracted widespread scientific interest. In the laboratory, various efforts have been made to produce synthetic sling filaments from the components of the spiroprotein precursor; However, replicating the mechanical properties of natural fibers remains a formidable challenge. This is perhaps not surprising, since the performance of Lasso silk stems from its complex hierarchical substructure, and to achieve this, the spider employs a complex mechanism that is coordinated by conformational changes in the domain of modular spirals, in response to precisely timed chemical triggers, combined with physical forces generated within the finite geometry of the filamentous rotating pipes.
The classic method of rayon spinning often involves the conversion of unnatural protein feedstock into fibrous material through harsh denaturing conditions, such as alcohol coagulation baths in wet spinning. Recently, however, there has been a growing interest in applying biomimetic technology to produce fibers with similar natural structures and functions under ecologically sound conditions. Here, the focus is on replicating the subtle biochemical processes that work together to allow silk fibers to self-assemble in their natural systems. These events include liquid-liquid phase separation (LLPS), which is considered a critical step in the preassembly of multiple protein fibers, and in spirochetes, it is regulated by repeat domains (RPDs) and C-terminal domains (CTDs) in response to gradients of kosmotropic ions, such as phosphate. At the same time, the acidification gradient mediates rapid end-to-end multimerization of helix protein chains and unfolding of CTDs via the N-terminal domain (NTD). Rheological effects such as shear and elongation flow are also major considerations, as they promote the gradual arrangement of helical protein chains within the spinning pipeline and initiate the transition of a wide range of RPDs from a largely disordered state to ordered intermolecular interactions, enabling the formation of dispersed β-sheet nanocrystals, which give the fibers higher strength.
Microfluidic Technology Microfluidic Technology Expert - Suzhou Wenhao Microfluidic Technology Co., Ltd. presents an exciting frontier in the assembly of structural proteins, in which complex macrostructures can be constructed, at least in theory, to introduce relevant physiological triggers in a flexible manner through customized channel arrangement and desired temporal sequence. In fact, for rayon spinning, microfluidic-based methods are considered to have the greatest potential to facilitate the assembly of similar natural fibers than more traditional techniques. In particular, the limited size and geometry of the rotating tubing determine the critical flow parameters, which can be approximated in the design of the miniaturized channel in the chip.
Previous studies have reported the incorporation of microfluidic principles in spinning artificial spider silk. In terms of biomimetic fidelity, these methods include the externalization of various recombinant helix protein sequences, microfluidic chip design, chemically triggered components, and the critical processing steps applied. A notable example shows the formation of fibers using a three-domain ADF3 spirochete by successive introduction of phosphate ions and pH reduction in a microfluidic device. However, crucially, previous studies have not reported on the production of fibers with multi-level nanoscale substructures, which are hallmarks of natural silk self-assembly. Supplementary Table 1 summarizes the differences in microfluidic spinning of recombinant spirochetes. Interestingly, for the spinning of regenerated silk fibroin (RSF), the forces generated inside the microfluidic chip have been quantified to show the distribution of shear and elongation along the channel, revealing the relationship between these forces and the contents of the β sheet. However, in the case of recombinant spirochetes, this quantitative analysis of the forces within the microfluidic channels has not been performed to correlate the forces with the formation of fibers and changes in conformation. Recombinant spirochetes in their native state are more responsive to naturally occurring triggers that lead to self-assembly behavior than RSF. Therefore, quantifying the shear stress and other forces under similar natural conditions is of great significance for better understanding the self-assembly mechanism of spider silk.
Here, we propose a rationally designed microfluidic system designed to simulate the functionality of a natural silk spinning device by integrating the latest insights into the self-assembly process in vivo. By successive application of triggered phase separation and biomimetic chemical gradients for nanofiber formation, as well as through quantifiable shear effects, we demonstrated the complete in-situ assembly of multihierarchical structural filament fibers from recombinant MaSp2 precursors with tunable β abundance and near-instantaneous velocity.
Electronic logo for a bionic microfluidic device
We aimed to design a biomimetic microfluidic device that approximates the natural chemical and physical gradients within the channel for studying spider silk fiber assembly. The architecture of the chip is shown in Figure 1a. It consists of 3 inlets, namely (1) a precursor spirochetal solution, (2) kosmotropic ions at neutral pH, and (3) a low pH fluttering trigger. The architecture provides two sequential mixing zones followed by an extended channel terminating at the egress, and defines three zones along the device for monitoring the sequential stages of self-assembly (Parts A, B, and C). The design is rationalized as follows: at the beginning of the flow, the merging of the flows originating from (1) and (2) exposes the spirochetes to a kosmotropic anion gradient similar to that found in spider spinning ducts, which is expected to trigger the LLPS of MaSp2 and the nascent protein droplets are transported downstream via section A via laminar flow. Subsequent contact with the flow originating from (3) in the opposite direction creates a pH gradient that mimics the natural acidification effect and is expected to trigger the solidification of the spirochetal condensate into a fibrous network (Section B). Part C constitutes a straight channel with a size of 2 cm×80 μm×62 μm, and mechanical deformation is expected to facilitate the assembly of elongated filamentous fibers.
To clarify, we have chosen a simplified design in which each individual channel has a uniform cross-section and the width is increased from B (60 μm) to C (80 μm) in order to focus on the effects of shear in regulating fiber assembly and crystallization while minimizing the effects of tensile flow. This is in contrast to the more complex (i.e., convergent) geometries found in natural spinning pipes, where the gradual narrowing of the pipe towards the spinneret creates a strong tensile flow that aids in the arrangement of the silk molecules. It is important to note that the sample flow in our system is initiated by applying negative pressure (vacuum) from the outlet, which was uncommon in previous filamentous fiber assembly strategies, where the flow is typically generated by positive pressure or syringe pumping techniques; This is to mimic the observation that natural silk fibers are "pulled" from the organism rather than being "pushed" (squeezed); In addition, this enables fine-tuned control of the forces generated within the microfluidic device.
From LLPS to nanofibrillation to fiber-forming native filament-like assembly
As a spider silk building unit, we used recombinant MaSp2 slingolea with a series of domain structures (Figure 1b), including the complete constructs of N-R6-C and N-R12-C (with 6 and 12 tandem repeats, respectively), the truncated constructs N-R6-x, x-R6-C, and x-R6-x, and the three-domain N-R12-C(xA) variant of the polyalanine block replaced with alternating glycine and serine residues (GSGSGSGSG), To assess the order dependence of β sheet formation during fiber assembly. The prepared spirochetes were confirmed by SDS-PAGE.
Outside of microfluidic systems, tridomain MaSp2 can be easily induced to form LLPS droplets by exposing Cos-level ions at neutral pH (Figure 1c), whereas under acidic conditions (pH 5.5-5.0), the combined effect of LLPS and NTD-mediated multimerization produces spontaneous and rapid network assembly (Figure 1c). In this study, we used a mixed citrate-phosphate buffered saline (CPB) system as a general kosmotropic trigger because of its improved buffering capacity within the relevant pH range (7.5–5.0) compared to individual phosphate and citrate components.
The stepwise biomimetic process is integrated into the microfluidic system and optimized to generate layered MaSp2 fibers with minimal intervention. The method itself simply requires the sequential addition of components at inlets 3, 2 and 1 at constant negative pressure and then immediately stopping the pump. The conditions for effective fiber formation are empirically established, which we define as the generation of a single continuous fiber within a microfluidic channel, excluding fiber breakage or the formation of aggregates or mixtures thereof. Here, it is critical to discover the composition of the fibrillation buffer, with the best results obtained for the CPB at concentrations between 1-1.5 M and pH 5.0, as well as the magnitude of the applied pressure and the magnitude of the applied pressure.
Figure 2a summarizes the results obtained using the labeled construct N-R12-C at −90 kPa pressure at 50 mg/ml (Inlet 1), 50 mM CPB pH 7.0 (Inlet 2), and 1.0 M CPB pH 5.0 (Inlet 3). A condensed MaSp2 structure can be observed over the entire channel length, with a clear evolution of the mesoscale morphology through successive phases of the device. Along section A, the channel wall on the side of Inlet 1 is densely covered with roughly spherical or amorphous deposits of MaSp2 condensate, with partial droplet fusion and surface wetting properties, consistent with the formation of LLPS and the downstream migration of protein droplets through laminar flow (Figure 2a-c). This result is consistent with the dynamic formation and fusion behavior of the droplet, as shown in Figure 1c. Although the surface contact between the buffer phase and the laminar flow trigger is relatively limited, the fact that MaSp2 is capable of a wide range of LLPS suggests that the initial conditions are close to the phase separation boundary, which ensures rapid phase separation when the two streams come into contact.
In segment B, the MaSp2 condensed water transforms into a flattened sheet morphology with a particle surface, which is an abrupt change triggered by the combined effect of the acidification gradient, which triggers fibrosis, and the shear effect caused by the change in channel geometry. In addition to the obvious changes in Part B morphology, the corresponding structure of spirochetes is influenced by the chemical and physical triggers introduced by this fibrosis stage (see below). Further downstream, in section C, we see the formation of continuous uninterrupted fibers over the entire length of the channel, with a circle diameter of 5-10 μm. Here, the layered substructure of the fibers is very pronounced, consisting of densely packed nanofibrils oriented along the longitudinal axis (Fig. 2d,e; Supplementary Film 2), which validates the bionic self-assembly strategy. Indeed, the aligned nanofiber bundles formed in section C of the device can be thought of as similar to the assembly of random protein network structures outside the device when mixing MaSp2 with CPB at pH 5 (Figure 1c), with the directional flow within the channel providing an additional factor of fibril arrangement; In other words, the 2D network structure can be converted into 1D nanofibers after the uniaxial orientation of the protein network. Once formed, the fibers can be recovered from the device for further analysis. Notably, the fibers were able to maintain structural integrity in pure water and exhibit high flexibility (Figure 2F). In some localized areas, the overall fibrous structure does rupture, which clearly reveals an underlying substructure consisting of individual protein fibrils with high aspect ratios, and is oriented along the longitudinal axis of the fibers in Figure 2g. Visualization of the entire length of the microfluidic device after assembly of the construct N-R12-C fibers is shown in Supplemental Film 3, which provides further evidence of hierarchical organization.
Interestingly, while the intact structures (i.e., N-R6-C and N-R12-C) produced fibers with microscopic indistinguishable morphology, the truncated variants N-R6-x, x-R6-C, and x-R6-x failed to produce any fibers under the same conditions, highlighting the critical interactions between different domains for successful self-assembly of spider silk under native conditions. In addition, while the three-domain N-R12-C (xA) lacking polyalanine block exhibits a similar morphology to the N-R6-C and N-R12-C structures in segments A and B, the fibrous structures produced by segment C often ruptures within the channel, reflecting the obvious mechanical weakness of these structures.
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