A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.
The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.
Spider and silkworm can produce outstanding silk fiber from aqueous protein solution at ambient temperature and pressure. Shearing and extensional flow can induce the formation of liquid crystal texture in the silk gland1. In recent years, there has been a great interest in mimicking the spinning process of the spider in order to produce high strength artificial fibers. However, large quantities of spider silk protein cannot be produced efficiently and economically by farming spiders due to cannibalism. Substantial amounts of silkworm silk can be obtained easily by farming. Otherwise, the silkworm and spider have a similar spinning process and amino acid composition. Therefore, silkworm silk fibroin is selected as a substitute to spin artificial animal silk by many researchers.
Spider and silkworm extrude protein solution through their spinning duct into fiber in air. The high stress forces generated along the spinning duct most likely stretch the silk fibroin molecules to a more extended conformation2. Artificial silk fibers have been spun using conventional wet spinning and dry-spinning processes3,4, which do not take into account the fluid forces generated in the spinning duct.
First, microfluidic approaches were used to investigate the assembly of silk protein5,6. Then, microfluidic fabrication of RSF was studied via modeling the shearing and extensional forces7,8. Young's modulus and diameter of RSF fibers can be tuned by microfluidic wet spinning, but the tensile strength of drawn fiber was less than 100 MPa7. Finally, high strength RSF fibers were successfully prepared using the microfluidic dry-spinning method, but the diameter of the fiber is only 2 µm8. Recently, microfluidic wet spinning was successfully used in the production of high strength recombinant spider silk fiber. The post-spinning drawing in air improved the surface and internal defects of artificial fiber9.
In this study, the improved microfluidic spinning process for RSF fiber is introduced. It aims to mimic the spinning process of silkworm silk, including the spinning dope, shearing forces, and dry-spinning process. This spinning method not only can produce high strength artificial silk fiber, but also can adjust the diameter of the fiber. Firstly, the RSF spinning dope was sheared and elongated in a biomimic channel with a second order exponential decay. Secondly, the influences of relative humidity (RH) on the fiber morphology and properties were studied in the microfluidic dry-spinning process10. Compared to the conventional spinning spinneret, our microfluidic system is highly biomimetic and can be used to produce high strength fiber from solutions at ambient temperature by the dry or wet spinning method.
Due to the high-resolution, high-brightness, and high-energy of the synchrotron radiation microfocus X-ray, it can be used to characterize the microstructure of a single fiber with a diameter of several micrometers4,11,12,13,14. Here, SR-WAXD technique was used to calculate the crystallinity, crystallite size and crystalline orientation of RSF fibers.
CAUTION: Please consult all relevant material safety data sheets before use. Several of the chemicals used in preparing the molding are acutely toxic. Please use personal protective equipment (safety glasses, gloves, lab coat, full length pants, and closed-toe shoes).
1. Microfluidic Spinning of RSF Aqueous Solution
2. Synchrotron Radiation Characterization of Crystalline Structure of RSF Fiber
High strength RSF fibers were successfully produced by using the microfluidic spinning method. The stress-strain curves and SEM images of the stretched RSF fibers C44R40 are shown in Figure 2. At least 10 fibers were measured in the tensile test. Stress-strain curves were chosen according to the average value of the breaking stress and strain of fibers. The WAXD data of the fibers are shown in Figure 3. The crystallinity and crystalline orientation were calculated according to the WAXD data. For sample designation, we use C and R to present the concentration of RSF in the spinning dope and the relative humidity, respectively. For example, the fibers spun from 44 wt% RSF spinning dope at 40 ± 5% RH was designated as C44R40, which was post-drawn at a draw ratio of 4. Other fibers were renamed as C44R50, C47R40, and C47R50 according to the same description.
Figure 1: Schematic of the fiber production and structure characterization. (a) Preparation of RSF solution, (b) microfluidic spinning process of RSF fibers, (c) synchrotron radiation experimental setup of RSF single fiber. Please click here to view a larger version of this figure.
Figure 2: Stress-strain curves of post-treated RSF fibers. Insert shows SEM image of C44R40. Scale bar = 10 µm. This figure has been modified from10. Please click here to view a larger version of this figure.
Figure 3: SR-WAXD data of post-treated RSF fibers. (A) Two dimensional WAXD patterns of post-treated RSF single fibers: (a) C44R40, (b) C44R50, (c) C47R40, (d) C47R50, and (B) degummed B. mori silk; (C) One dimensional WAXD data of post-treated RSF fibers and degummed B. mori silk, which was performed at peak deconvolution in (D). This figure has been modified from reference10. Please click here to view a larger version of this figure.
During the dialysis of the RSF solution, the pH value is critical for the following concentration process. If the pH value of the deionized water is smaller than 6, the RSF solution will be easier to gel during the concentration process. To avoid gelation, CaCl2 is added to the RSF solution. The concentration of CaCl2 is 1 mmol per weight of RSF.
Our previous work demonstrated the possibility of microfluidic dry-spinning of an RSF aqueous solution8. The geometry of the microfluidic channel was a simplified single-stage exponential function. For spider and silkworm, spinning dopes were draw down through a two-stage exponential regression spinning duct before fiber formation1,20. Here, the geometry of the microfluidic channel was designed by mimicking the second order exponential decay function of the silkworm spinning duct1. The width of the microfluidic channel decreases from an initial width of 2,065 µm to the terminal width of 265 µm, and the length of the elongation channel is 21.5 mm. In the previous article, the diameter of the drawn RSF fiber was 2 µm. Thus, a bundle of RSF threads had to be used for the mechanical test and structure characterization8.
The experiment shows that RSF concentration and relative humidity affect the diameter and microstructure of the RSF fibers in the dry spinning process. The RSF fiber spun at 40% RH shows a larger diameter and more crystalline structures than the fiber spun at 50% RH. However, the fiber spun at 50% RH has a higher crystalline orientation than that spun at 40% RH. The results might be related to evaporation rates of water at different humidities. A higher evaporation rate of water at 40% RH improves the intramolecular interactions and facilitates the fast phase transition of silk fibroin from sol-gel to solid silk fiber. A lower evaporation rate of water at 50% RH leads to a higher content of residue water in the solidified fiber. As a small molecular lubricant, the water facilitates the silk fibroin orientation and makes the partially solidified fiber to be stretched to finer fibers. This process helps us understand how water influences the formation of silk fibers during native spinning process.
The mechanical properties of post-treated RSF fibers are better than those of degummed silks4. After post-treatment, crystallinities of the fibers were drastically increased. The FWHM of the post-treated RSF fiber is smaller than that of as-spun fiber. It indicates that post-treatment improves the orientation of crystallites along the fiber axis. However, the complexity of the post-treatment process limits the mass production of RSF fibers with high strength.
Compared to a conventional spinneret, the microfluidic channel is well suited to mimic the geometry of a natural silk gland. Meanwhile, the microfluidic spinning was used to produce recombinant spider silk with outstanding mechanical properties9. Shearing and elongational sections were integrated in the microfluidic spinning chip to induce assembly and orientation of the protein molecules and fibrils. Therefore, the microfluidic spinning is promising in the production of high-performance animal silks, as well as other synthetic fibers from solution. However, the microfluidic spinning method can only produce single filaments and it cannot afford high production of artificial fibers.
The authors have nothing to disclose.
This work is sponsored by the National Natural Science Foundation of China (21674018), the National Key Research and Development Program of China (2016YFA0201702 /2016YFA0201700), and the “Shuguang Program” supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (15SG30), DHU Distinguished Young Professor Program (A201302), the Fundamental Research Funds for the Central Universities, and the 111 Project (No.111-2-04).
B. mori Cocoons | Farmer in Tongxiang, Zhejiang Province, China | ||
Sodium carbonate, anhydrous, 99.8% | Shanghai Lingfeng Chemical Reagent Co., Ltd., China | Analytically Pure | |
Lithium bromide, 99.1% | Shanghai China Lithium Industrial Co., Ltd., China | Analytically Pure | |
Calcium chloride, anhydrous, 96.0% | Shanghai Lingfeng Chemical Reagent Co., Ltd., China | Analytically Pure | |
Ethanol, anhydrous, 99.7% | Sinopharm Group Chemical Reagent Co.,Ltd., China | 10009218 | Analytically Pure |
SU-8 photoresist | MicroChem Corp., USA | ||
Developing solution | MicroChem Corp., USA | ||
Sylgard 184 | Dow Corning, USA | ||
Isopropanol | Shanghai Lingfeng Chemical Reagent Co., Ltd., China | Analytically Pure | |
Concentrated sulfuric acid | Pinghu Chemical Reagent Factory, China | Analytically Pure | |
30 vol% hydrogen peroxide | Shanghai Jinlu Chemical reagent Co., Ltd., China | Analytically Pure | |
Acetone | Shanghai Zhengxing Chemical Reagent Factory, China | Analytically Pure | |
Oxygen plasma treatment | DT-01, Suzhou Omega Machinery Electronic Technology Co., Ltd., China | ||
Syringe pump | KD Scientific, USA | KDS 200P | |
Humidifier | SEN electric | ||
Driller | Hangzhou Bo Yang Machinery Co., Ltd., China | bench drilling machine Z406c | |
Material testing system | Instron, USA | Model: 5565 | |
PeakFit | Systat Software, Inc., USA | Version 4.12 |