Biomimetic Spinning Of Regenerated Silk Fibroin Aqueous Solutions Using Microfluidic Chips

Natural animal silks from spider and silkworm have many remarkable properties. Especially for dragline silk from Nephila clavipes spider, it is much tougher than Kevlar and carbon fiber, and is even one of the toughest fibers in the world. Furthermore, spider and silkworm use an environmentally friendly dry spinning process to produce silks from all aqueous solutions at ambient temperature and pressure. In order to obtain the artificial animal silks and mimic the low-carbon green spinning process, wet spinning, electrospinning and dry spinning techniques have been taken to produce artificial silk fibers from regenerated and recombinant silk protein solutions by partially mimicking the natural spinning process. Nevertheless, the in vitro modelling has long been hampered for the low strength of artificial silk fibers. The reason is that the current biomimetic engineering grossly oversimplified the mechanisms by which natural fibers are formed. When spinning dope flows through the slender glands and spinning ducts in silkworm and spider, the protein concentration of the spinning dope increases, while other contents of the dope, such as metal ion concentrations and pH values, are also dynamically adjusted through the surrounding secretory cells. At the same time, the actions of the shearing and elongation on the protein aqueous solution are also performed during the spinning process. Unfortunately, it is not possible to reproduce the dynamic character and the complexity of the in vivo process using equipments in traditional laboratories.The development of microfluidic technology inspires its application in spinning and even biomimetic spinning of natural animal silk proteins. In addition, the diameter of the natural spinning duct dropped from107to10μm for spiders, and from396to54μm for silkworms. Thus, the spinning systems in vivo can be considered as typical microfluidic devices, in consideration of the structure and multifunction of the spinning apparatus. Consequently, microfluidic technology might be a perfect candidate to mimic the flow conditions and the multifunction of the spinning apparatus of spiders and silkworms. In this thesis, various microfluidic chips were designed to respectively mimic the functions of the natural spinning systems. Artificial fibers were also prepared from regenerated silk fibroin (RSF) aqueous solution using the biomimetic chip.Firstly, microfluidic chips were prepared based on photolithography and rapid molding technology. The fabrication process was analyzed and solved to determine the fabrication parameters. By biomimicking the glands and the spinning ducts of silkworms and spiders, a microfluidic channel in a micro-chip was designed and fabricated. The chip was used for pH or Ca2+concentration dynamically adjustments of RSF aqueous solution. Due to the laminar flow characteristic of microfluidics, RSF aqueous solution and a buffer (pH or Ca2+) tended to keep their laminar streams without mixing. Thus, the RSF concentration was maintained consequently, while the pH value or Ca2+concentration of the RSF aqueous solution was dynamically adjusted via ionic diffusion during the RSF solution flowing in the microfluidic channels. The results showed that it was easier to adjust the pH value of the RSF aqueous solution to the target pH value of the buffer, when the two streams had closer pH values. In addition, The lower flow rates of the two streams and the decreasing RSF concentration of RSF aqueous solution improved the pH value and Ca2+concentration dynamically adjustment of the RSF aqueous solution.By biomimicking the dynamical enrichment of the spinning process in vivo, two-layer microfluidic concentrators were designed and used for the enrichment of RSF aqueous solution by microdialysis. In order to choose appropriate dialysis membrane and water absorbent. Cellulose dialysis tubes containing RSF aqueous solutions were initially immersed respectively in different beakers containing polyethylene glycol (PEG) aqueous solution. Results showed that PEG aqueous solution could be used to enrich the RSF aqueous solution in cellulose dialysis tubes, and the enrichment effect was improved after extended immersing time. The microfluidic devices based on regenerated cellulose membrane involved a flow of RSF aqueous solution on a donor side and a flow of PEG aqueous solution on an acceptor side. The results showed that the chips could be used as micro-concentrators for RSF aqueous solution enrichment. The RSF concentration of the solution could be enriched up to31.2wt%from12wt%by using the microfluidic chip. The enrichment efficiency increased with the increasing acceptor concentration, increasing channel length, depth and decreasing flow rate of RSF aqueous solution. However, the enrichment efficiency increased with the increasing PEG aqueous solution flow rate up to certain value.In addition to the mimic of content adjustment of RSF aqueous solution, the dimension of spinning apparatus in vivo was also biomimicked in microfluidic chips with a single channel. Raman spectroscopy and small angle x-ray scattering (SAXS) technique were adopted to analyze the conformation transition of RSF and the shape parameters of RSF aggregates in RSF aqueous solutions before and after flowing in the microchannel. Results showed that higher RSF concentration, lower pH value (pH4.8) and higher Ca2+concentration promoted the growth of RSF aggregates and the sensitivity of RSF aqueous solution to the elongation and shearing condition. When the concentration of RSF and Ca2+increased, or pH value decreased (pH4.8), the transition from the a-helix structure to P-sheet structure in RSF aqueous solution became more obvious at the same elongation and shearing conditions in microchannels. Moreover, when the elongation rate and the shearing time in the microchannels were respectively varied, a proper increase of β-sheet structure of RSF in the RSF aqueous solution (40wt%, pH4.8, c(Ca2+)=0.3M) was observed with increasing elongation rate or shear time in the microchannels. The results indicated that the increasing shear time and elongation rate had similar effects on the structure transition of RSF in aqueous solution.Based on the above studies, the microfluidic chips were also used to dry spin RSF fibers from highly concentrated aqueous solution (50wt%, pH4.8, c(Ca2+)=0.3M). The microfluidic channels with various geometries as exponential function were adopted to mimic the natural elongation and shearing condition. It was found that the conformation transition of silk fibroin and the crystalline structure in RSF as-spun fibers were improved, when the elongation rate, the elongation time, and the shearing rate increased. Thus, the mechanical properties of the as-spun RSF fibers were enhanced. To improve the structure and mechanical properties of the RSF fibers, they were further post-treated. It was obvious that the post-treatment played a significant role in the formation of β-sheet structure and the improvement of silk II crystalline structure. The post-treated RSF fibers showed excellent mechanical properties including a Young’s modulus of19GPa, a breaking stress of614MPa, and an exceptional breaking strain of27%. They are superior to those of the degummed cocoon silk, though they cannot still rival the corresponding properties of Nephila clavipes spider dragline silk. Due to the low elongation rate of microchannels in these experiments, the variation of the elongation rate did not play an important role in the structure and mechanical properties of RSF as-spun fibers and post-treated fibers. Compared with the variation of the elongation rate, the extension of shearing time in microchannels obviously improved the structure and mechanical properties of RSF as-spun fibers and post-treated fibers. A dramatic increase of ordered structure was also observed in RSF post-treated fibers with an increasing shearing time in the microchannels. At last, the pH unadjusted RSF aqueous solution (50wt%, pH7.8, c(Ca2+)=0.3M) was also used as the spinning dope to dry spin RSF fibers in microfluidic channels with various geometries. It was found that the dimension of the microchannel played an important role in the spinnability of the RSF aqueous solution in the present studies. When the outlet of the channel is wide, it was easy to continuously obtain fiber with certain mechanical properties. However, the present chip with narrow outlet still cannot be used to spin fiber at present and need further investigation.