A Premier Study On Bomimicking Spinning For Concentrated Regenerated Silk Fibroin Aqueous Solution

In this thesis, the development of biomimetic spider and silkworm silk has been briefly introduced. The processes and properties of resultant man-made silk fiber have also been summarized. In our work, silk fibroin of silkworm is selected as a model system because there is still not enough spider silk protein that can be supplied to do spinning and on the other side, the composition and conformation of silkworm fibroin is very similar to spider protein.Concentrated regenerated silk fibroin （RSF） aqueous solutions with concentration close to that in the gland of silkworm were prepared. The effects of storage time and concentration on the conformational transition of the concentrated RSF aqueous solution were studied by Raman spectroscopy and circular dichroism （CD） spectroscopy. The effects of concentration, shearing, pH values and adding surfactant on rheological properties and conformational transition of RSF aqueous solutions with high concentration were also studied. Besides, the regenerated silk fibers were obtained from the RSF aqueous solution with different spinning conditions by so called "capillary （like the gland of silkworm） spinning processes "at ambient temperature. The orientation of these fibers was measured by polarizing microscope. Their secondary structure was investigated by Raman spectroscopy and related mechanical properties were also measured.The effect of storage time and concentration on the conformational transition of the concentrated RSF aqueous solution （15.5%, 25.5% and 31%） was studied by Raman spectroscopy and circular dichroism （CD） spectroscopy. It was suggested that the conformation of silk fibroin in concentrated RSF aqueous solution is unstable at ambient temperature. The storage time played an important role in the conformational transition of RSF aqueous solution. Even if the concentration was kept at constant, the conformation of silk fibroin was transformed automatically from random coil/α-helix toβ-sheet structure with the increasing of storage time. And the conformational transformation was accelerated with the increasing of concentration. At the same time, from Raman and CD spectra analysis, it was found that theβ-sheet structure would increase as the concentration increased. In addition, the storage time and concentration of the RSF solution have some relationship. The higher the concentration of RSF aqueous solution was, the shorter the storage time of solution should be. This work indicated, in the biomimicking spinning process of silkworm, the storage time of fresh RSF aqueous solution should be considered together with fluidity of RSF aqueous solution. The investigation implied that the original dilute RSF aqueous solution should be stored under low temperature and concentrated just before spinning.The shearing has greatly effect on the formation of regenerated silk. The thermal properties of original and sheared concentrated RSF aqueous solutions from 15% to 37% were investigated by differential scanning calorimetry （DSC）. And the effect of shear rate (1000 s^-1and 2000 s^-1) on the conformation of concentrated RSF aqueous solutions was studied by Raman spectroscopy. It was discovered that the endothermic peaks of all RSF aqueous solution in the DSC curves shifted toward the lower temperature region after shearing. The original and sheared solutions with concentration less than 27% exhibit a prominent single endothermic peak in the DSC curves. However, when the solution of concentration excess 30%, the DSC curves display two major endothermic peaks. Under the action of stress, the conformation of proteins in the concentrated RSF aqueous solution can be changed from random coil andα-helix toβ-sheet conformation. And the conformational transformation was accelerated with the increasing of shearing. For the 37% RSF aqueous solution, an irregular insoluble material with P-sheet conformation could be found after applying the shear strain of 2000 s¹. These results confirmed that the shearing is a key parameter in determining the structure of silk fibroin. But the match of concentration and shear stress are crucial in the spinning process of RSF aqueous solution..When silkworm is spinning, the silk fibroin aqueous solution is subjected to both shearing and drawing. In order to mimic the spinning process of silkworm, the shearing force was applied by pressing the RSF aqueous solution through a capillary, like the gland of silkworm. And then the RSF aqueous solution was drawn by external force. The regenerated silk fibers can be obtained from concentrated RSF aqueous solution without any organic solvents being used and the fiber is spun into air at ambient temperature just like the silkworm did. The birefringence of the samples showed that the degree of orientation could be increased by shearing. From Raman spectra analysis and mechanical properties measurement, it also can be concluded that the shearing is important for increasingβ-sheet structure and improving mechanical properties of the regenerated silk fibers. However, such regenerated silks obtained from the aqueous solution without adjusting pH value, type and concentration of ion displayed poor mechanical properties than that of natural silkworm silk. To improve the mechanical properties of regenerated silk fibers, it is necessary to make RSF aqueous solutions have the same properties and structure of natural fibroin solution.The RSF aqueous solutions with high concentration were alkalescence with a pH of around 8.5, different from the pH （less than 7） of silk fibroin solution in the silkworm’s gland. Therefore, RSF aqueous solution was adjusted to the same pH values （pH=6.9, 5.6, 4.8） as the posterior、middle and anterior division of the silkworm’s gland using citric acid buffer solution. It was found that the viscosity increased with the reducing of pH values of RSF aqueous solution with the same concentration. The same results could be obtained for spinnability of RSF aqueous solution with different pH values. That is to say, the spinnability could be improved by the reducing of pH value. The regenerated silk fibers were produced by capillary dry spinning technique at ambient conditions from RSF aqueous solution with different pH values. It was showed that the degree of orientation and mechanical properties of regenerated silk could be increased by adjusting pH values to acid solution. From Raman spectra analysis, it also can be concluded that the formation ofβ-sheet was accelerated by reducing pH value of solution, and the acidic solution is more favorable for conformational transition from random coil and a-helix to B-sheet conformation. Sericin play an important role during the formation of silkworm silk. In this paper, we substituted polyethylene oxide （PEO） for the sericin, and prepared the fibroin/PEO blend aqueous solution with different PEO content. The results showed that adding PEO could decrease the surface tension of RSF aqueous solution, and the decreasing of surface tension becomes more and more obvious with the increasing of content of PEO. Similarly, with the same concentration, the viscosity and spinnability of the solution were improved by adding PEO. And a better result could be obtained with the increasing of content of PEO. The regenerated silk fibers could be produced by capillary dry spinning technique at ambient conditions from RSF aqueous solution with different PEO content. It was found that the lowest spinnable concentration decreased with adding PEO. However, the effect of adding PEO on the degree of orientation、conformation and mechanical properties of regenerated silk was not obviously. In order to get a high performance regenerated silk, the techniques of mimicking the spinning process should be further developed.