| As one of the toughest materials, spider dragline silk exhibits exceptional strength and toughness that rival those of most of synthetic high performance fibers. Spiders use renewable materials as input and water as a solvent. Moreover, they produce fibers in air at room temperature and ambient pressure. The whole process is environmentally friendly. The outstanding properties of spider dragline silk are determined both by its chemical composition and the spinning process. However, it is not possible to obtain industrially useful quantities of spider silk by farming spiders, because spiders eat each other. Consequently, there has been a wide spread interest in biomimicking spider silk artificially, which leeda to a decade of wave to obtain recombinant spider silk proteins by using genetic engineering. Unfortunately, this method still suffers some problems. Firstly, the molecular weights (MWs) of most of recombinant spider silk proteins are only about one-third or less of those of the natural spider silk proteins. Secondly, gene vectors exclude the heterologous genes and lead to protein denaturation. As a result, the level of silk protein expression in heterologous systems to date has been universally low. Thirdly, the cost of the recombinant spider silk proteins is too high for even lab use at the moment.Since no enough raw or recombinant spider dragline silk protein can be supplied for biospinning, it is necessary to find an appropriate resource to investigate the biospinning conditions. In place of spider silk, Bombyx mori (B. mori) silk fibroin is selected as a model system to artificially spin silk fiber by many researchers. This is attributed to the similar spinning mechanism between silkworm and spider, and the similar amino acid composition between B. mori silk fibroin and spider dragline silk protein. Immobilized silkworm under steady and controlled conditions even could produce fibers with comparable mechanical properties as spider silk. In addition, unlike spider silk, substantial amounts of silkworm silk can be obtained easily by raising silkworm. Therefore, in this thesis, regenerated silk fibroin (RSF) fibers were dry-spun from their aqueous solutions and the as-spun fibers were post-treated to improve their mechanical properties.In regeneration process, fibroin molecules degrade to some extent. Since MW of RSF plays a very important role in dope stability, spinnability and microstructure of fiber, it is necessary to determine the MW of silk fibroin after degradation. In this thesis, a simple non-gel sieving capillary electrophoresis (NGSCE) method was established to determine the MW of silk fibroin by using polyethylene glycol (PEG) 6000 as sieving matrixes. PEG 6000 could react with the capillary wall to reduce protein adsorption and form a network structure to separate the fibroin molecules. It was found that most RSF molecules we prepared had a wide molecular weight distribution (MWD) from 75.3 to 91.7 kDa, mainly at 83.8 kDa, while low content molecules distributed at 32.7 and 58.5 kDa. NGSCE showed a similar MWD with that obtained from the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and presented a higher sensitivity to small content of molecules than SDS-PAGE. The background electrolyte was composed of the three components, PEG, tris(hydroxymethyl)aminomethane (Tris) and sodium dodecyl sulfate (SDS). NGSCE showed a good linear relationship with fine reproducibility between the migration time and the MW of three standard proteins in the same background electrolyte. It was found that the composition of background electrolyte changed slowly with time. It was necessary to replace the buffer solution weekly and re-establish the calibration curve. The calibration curve must also be re-established whenever the capillary column was replaced. The NGSCE analytical approach requires very little sample preparation and provides fast results and good reproducibility and accuracy. These features render this method a powerful tool to determine the MW of RSF.A custom-built capillary spinning equipment was applied to spin fibers from RSF aqueous solutions in air by using a dry spinning process like those of silkworm and spider. Unlike wet spinning process, which is widely used in artificial spinning of animal silks, the dry-spinning process is environmentally friendly and low-cost. Compared to the traditional air pressure dry-spinning equipment, the new one has simple structure, and requires less volumes of dope. Moreover, RSF fibers could be successfully prepared from various RSF aqueous solutions (pH=4.8-6.9, several kinds of ions) under a wide length diameter ratio (L/D) ranging from 40 to 133. The effects of MW, pH, metal ions, RSF concentration and spinning parameters on the spinnability of the spinning dopes were investigated from the rheology behaviors of the dopes. It was found that the MW of RSF ranged from 20～100 kDa, which was much smaller than that of natural silk fibroin. Moreover, too high MW deteriorated the spinnability of the dope in the present studies. Thus partial degraded fibroin allows generating stable and spinnable RSF solutions. After appropriate condensation, the dopes with a pH ranging from 4.8 to 6.9 (Ca2+concentration=0.3 mol/L) were successfully used to dry-spin fibers, which showed comparable spinnability and fibroin concentration. Rheology analysis showed that a reduction in pH induced an increase in the relaxation time of the dope. Different kind of metal ions affected the spinnability of the dopes. The dopes only adding Ca2+ had small relaxation times, good spinnability and smooth oscillatory sweep curves, while the dopes adding Mg2+, K+ showed opposite trends. The RSF concentration of spinnable dope ranged from 44.2% to 48.1%, which was much higher than that in B. mori spinning duct. When the RSF concentration was as low as 39.8%, the unspinnable dopes showed behaviors as viscoelastic liquid in the oscillatory frequency sweep. When the RSF concentration was as high as 55%, the dopes changed quickly from viscoelastic liquid to viscoelastic solid, and the spinnability was deteriorated. Our experiments showed that the spinnable RSF dopes had relaxation times smaller than 0.01 s. The smaller the relaxation time was, the better spinnability of the dopes had and vice versa. Therefore, the relaxation time could be used as a basic criterion of spinnability of the RSF dopes. Although the dry-spinning process is similar to those of silkworm and spider, there are still some differences. Firstly, the structures of RSF molecules are different from those of natural fibroin molecules; secondly, the process is not liquid crystalline spinning as silkworm and spider. Therefore, the as-spun fibers have very low orientation degree and low content of P-sheet conformation. Despite the mechanical properties of the as-spun fibers could be improved by increasing of L/D and take-up speed, the stress is still quite smaller than that of natural silk.In order to improve the mechanical properties of the as-spun fibers, a post-treated process was presented. Two post-treatment agents systems, alcohol/water system and inorganic salt system, were adopted to investigate the effects on the conformation and the mechanical properties of the fibers. It was found that alcohol/water system obviously induced the fibroin conformation transition from random coil/α-helix toβ-sheet and improved the mechanical properties of the fibers. For the inorganic salt system, only saturated ammonium sulfate ((NH4)2SO4) could render a weak conformation transition of RSF from random coil/α-helix toβ-sheet. Two methods were employed to post-treat the as-spun fibers. Compared to method A (the fibers were firstly immersed in post-treatment agent for 1 h, and then manually drawn with 2.0 draw ratio in steam), method B (the fibers were drawn 2 times in post-treatment agent, and then immersed in the solution for 1 h) was more effective in promoting the morphology, structure and mechanical properties of the RSF fibers. By using method B, the fibers treated in ethanol/water and isopropanol/water solutions showed very smooth surface as degummed silk, whereas the fibers treated in methanol/water solution and saturated (NH4)2SO4 solution exhibited uneven surface. Sample Et-D-I-B that was treated in ethanol/water solution by using method B showed the better performance in comparison with other post-treated fibers. The breaking strength of Et-D-I-B was improved to 199.2 MPa, which was still lower than that of degummed silk. However, the elongation at break of Et-D-I-B was improved to 55.4%, which was much higher than that of degummed silk. This indicates that the fiber has potential to be further stretched. The relatively low breaking strength is probably related to the microstructure defects in the RSF fiber, which has less developed crystalline regions and lower orientation than natural silk fiber. Unoriented amorphous molecules are also distributed among crystalline regions. On the contrary, silkworm produces silks using liquid crystalline spinning process, in which the molecules neatly arrange and somewhat orient before spinning. When the fiber is drawn 2 times in 80 vol%ethanol aqueous solutions, and then immersed in the solution for 1 h, the tensile strength and the elongation at break of the fiber increase effectively and might be further improved.In order to further improve the mechanical properties of the RSF fibers and explore the optimal post-treatment conditions, the effects of draw ratio and immersion time on the structure and mechanical properties of the RSF fibers were studied by choosing 80 vol% ethanol aqueous solutions as post-treatment agent. With the increase of draw ratio, the breaking strength and the orientation degree of the fibers increased. When the immersion time was set as 90 min, the fibers drawn 1-3x showed comparative contents ofβ-sheet conformation. The fibers firstly drew 3 times then immersed in 80 vol% ethanol aqueous for 1-120 min were also prepared. Their mechanical properties and the quantitative analyses of Fourier transform infrared spectroscopy (FTIR) showed quite similar trends. With the increase of immersion time, both the content ofβ-sheet conformation and the breaking strength of the RSF fibers increased sharply at beginning, then approached to a constant value after 90 min of immersion. The temporary optimal post-treatment process in this thesis is post-drawing the as-spun fibers 3 times in 80 vol% ethanol aqueous solutions, and then immersing the fiber in the solution for 90 min. The obtained fibers exhibited smooth surfaces, uniform diameter and larger elongation at break than that of degummed silk. The breaking strength of the RSF fibers is up to 301 MPa, which approached that of degummed silk. Despite the MW of RSF molecule were much lower than that of natural silk molecule, the breaking energy of RSF fiber was 2.4 to 3.8 times higher than that of degummed silk. It demonstrated that the determinant of the mechanical properties was secondary structure rather than molecular weight. Moreover, the dry-spun fibers from different RSF solutions with a pH of 4.8,5.2,5.6 and 6.0 were post-treated with a draw ratio of 3 and an immersion time of 90 min, respectively. The results indicated that the pH of the dopes had limited effects on the structure and mechanical properties of the post-treated fibers. The post-treated fibers from the dopes with different pH had similar morphology, structure and mechanical properties which all rivaled that of degummed silk. Therefore, the post-treatment process is suitable for all the as-spun fibers from the dopes with different pH. By using 80vol% alcohol solution as post-treated agent, this thesis could finally produce fibers with excellent performance. As a post-treated agent, the ethanol aqueous system is non-toxic, environmentally friendly, and hence has promising applications.
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