【作者】 魏伟；

【导师】 胡学超；

【作者基本信息】 东华大学 ， 高分子物理与化学， 2011， 博士

【摘要】 蜘蛛丝的力学性能比大部分的合成纤维更加优异,是迄今为止最强韧的材料之一。而且蜘蛛丝的生产工艺非常“低碳环保”,只需在常温常压下以水为溶剂在空气中直接成丝,整个过程所需原料也都是可再生的。蜘蛛丝优异的性能是由其化学结构和纺丝工艺共同决定的。然而,和蚕不同的是,由于蜘蛛同类相食无法大量饲养,所以无法通过天然的方法获得大量蜘蛛丝。因此人们一直对模仿蜘蛛的纺丝过程从而人工制备蜘蛛丝有着浓厚的兴趣。在过去的十几年中,全世界掀起了一股采用基因工程法制备重组蜘蛛丝蛋白的浪潮。然而该方法仍存在着几个难题。首先,重组蜘蛛丝蛋白的分子量太小,大多数只有天然蜘蛛丝蛋白分子量的三分之一甚至更少；其次,基因载体对外来蜘蛛丝蛋白基因产生排斥,会导致重组蜘蛛丝蛋白变性,从而使目前重组蜘蛛丝蛋白的水平仍然偏低；最后,基因工程蛋白成本太高,甚至无法满足实验室使用需求。由于没有足够的天然或重组的蜘蛛丝蛋白,因此必须找到一种适当的替代物来进行仿生纺丝的研究。最终很多学者选择蚕丝蛋白作为模型替代蜘蛛丝蛋白以进行仿生纺丝研究。这是因为蚕丝与蜘蛛丝的成丝过程比较相似,而且丝素蛋白与蜘蛛丝蛋白的氨基酸组成也比较接近。研究表明在一定条件下,蚕丝的机械性能甚至可与蜘蛛丝媲美。另外,与蜘蛛丝蛋白不同,人们可以非常容易地获得大量的蚕丝蛋白以满足研究需要。因此本论文以再生丝素蛋白水溶液为模型进行了干法纺丝的研究,并探索了后处理方法和条件以提高再生丝素蛋白纤维的力学性能。在再生过程中,丝素分子会发生一定程度的降解,且丝素分子的分子量对于再生纺丝液的稳定性、可纺性以及再生纤维微结构有较大的影响,因此准确测量降解后丝素蛋白的分子量是十分必要的。本论文开发了一种使用聚乙二醇(PEG)6000作为筛分介质的无胶筛分毛细管电泳法(NGSCE)以测量再生丝素蛋白的分子量。PEG 6000可以降低丝素蛋白对毛细管壁的吸附,并在毛细管内互相缠绕形成网状结构,从而对丝素蛋白分子进行筛分。测试结果表明,采用本方法制备的再生丝素蛋白中少量丝素蛋白分子量分布在32.7和58.5 kDa附近,大部分分子的分子量在75.3至91.7 kDa的范围内,其中83.8 kDa附近含量最高。该结果与十二烷基硫酸钠聚丙烯酰胺凝胶电泳法(SDS-PAGE)测试得到的分子量分布结果是相似的,同时NGSCE法对于低含量分子也更加敏感。该方法的电泳缓冲液由PEG、三羟甲基氨基甲烷(Tris)和十二烷基硫酸钠(SDS)组成。同一电泳缓冲液下,三种标样蛋白的分子量和迁移时间呈现良好的线性关系和重复性。但是实验也发现电泳缓冲液会随着时间慢慢发生一些变化,有必要每周更换缓冲液并重新标定标准曲线。更换毛细管时也必须重新标定标准曲线。本方法需样量少、耗时少、重复性好,为研究仿生纺丝过程中分子量的影响,提供了方便手段。本论文采用与蚕和蜘蛛纺丝相一致的干法纺丝工艺,通过自制的毛细管纺丝装置从以水为溶剂的纺丝液中成功地制备了再生丝素蛋白纤维。与报道较多的湿法纺丝不同,本论文采用的干法纺丝更加环保而且成本更低。相比传统的气压式干法纺丝装置,新装置结构简单、所需纺丝液少,且适用于多种纺丝液(pH=4.8-6.9,多种金属离子)在更大的长径比(40-133)条件下进行纺丝。本论文还采用流变的方法研究了再生丝素蛋白的分子量、pH、金属离子、丝素浓度和纺丝条件对纺丝液可纺性的影响。实验发现：在本论文条件下,再生丝素蛋白分子量范围约为20-100 kDa,远小于天然丝素蛋白分子量。而且在本论文条件下,再生丝素蛋白的分子量太高会降低纺丝液的可纺性,适当地降低分子量有利于制备再生丝素蛋白纤维。通过适当的浓缩,pH在4.8-6.9范围内的纺丝液(Ca2+离子浓度均为0.3 mol/L)都可用成功制备再生丝素蛋白纤维,不同pH纺丝液的纺丝浓度和可纺性相差不大。纺丝液的流变测试结果表明,随着pH的降低,纺丝液的松弛时间增加。不同金属离子对再生纺丝液的可纺性影响很大,只添加Ca2+离子的再生丝素蛋白水溶液松弛时间较小,可纺性较好,动态流变曲线比较平滑；而添加Mg2+离子和K+离子的纺丝液松弛时间较大,可纺性较差,动态流变曲线波动较大。再生丝素蛋白水溶液最佳的可纺浓度为44.2-48.1%(用称重法测量),该范围大于蚕体内的可纺浓度。浓度低于39.8%的纺丝液在进行动态流变测试时始终呈现粘弹性液体状态,无法进行干法纺丝。而当浓度高于55%时,由其动态流变曲线可知,纺丝液在较低角频率条件下就易由粘弹性液体状态向粘弹性固体状态转变,从而使纺丝液的可纺性变差。根据大量实验得到的经验表明,所有可纺的再生丝素蛋白纺丝液的松弛时间均小于0.01s；在此范围内,松弛时间越小,溶液的可纺性越好；松弛时间越大,溶液的可纺性越差。松弛时间可作为简单判断纺丝液可纺与否的一种依据。尽管本文的干纺方法与蚕和蜘蛛的吐丝过程比较相似,但也有一些不同。首先再生丝素蛋白结构与天然丝素蛋白结构有所不同,其次目前的纺丝尚不是液晶纺丝,所以初生丝的取向度低,p-折叠构象含量也较低。尽管增加毛细管长径比或增大卷绕速度均可明显提高初生丝的力学性能,但是其断裂强度绝对值要远小于天然蚕丝。为了解决再生丝素蛋白初生丝力学性能不佳的问题,本论文对初生丝进行了后处理探索,试图通过后处理提高纤维的力学性能。在总结文献的基础上,本论文研究了两类后处理剂(醇-水溶液体系和无机盐水溶液体系)对再生丝素纤维构象和力学性能的影响。实验发现醇-水溶液体系可以显著地促使初生丝构象从无规卷曲/α-螺旋构象向β-折叠构象转变。而无机盐水溶液体系中只有硫酸铵可以微弱地促使初生丝产生这种构象转变。经过醇-水溶液体系处理的丝素蛋白纤维力学性能较大幅提高。本论文还研究了两种不同后处理方法对初生丝性能的影响。结果表明,相比于方法A(将初生丝在后处理剂中浸泡1h,然后在蒸气浴中拉伸2倍),方法B(将初生丝在后处理剂中拉伸2倍,然后再浸泡1h)能够更加有效的促进纤维形貌、结构和力学性能的改善。对乙醇-水和异丙醇-水体系而言,采用方法B处理过的纤维具有和脱胶丝—样的光滑表面,而对甲醇-水体系和硫酸铵水溶液而言,采用方法B处理过的纤维的表面非常褶皱。通过对比,样品Et-D-I-B(将初生丝在乙醇-水中拉伸2倍,然后再浸泡1h得到的纤维)的各种性能最佳,其断裂强度和断裂伸长率可以分别达到199.2 MPa和55.4%。尽管其断裂强度低于天然脱胶丝,但是其断裂伸长率高于脱胶丝。样品Et-D-I-B较高的断裂伸长率也表明其能够被进一步拉伸。本论文认为样品Et-D-I-B断裂强度比天然丝差可能是由于Et-D-I-B的结晶和取向结构没有天然蚕丝完善,晶区之间尚存在一定量的卷曲的无定形态的丝素蛋白分子未被拉伸。而蚕采用的是液晶纺丝,在液态时分子已排列整齐并有一定程度取向以后才开始纺丝,所以结晶结构完善、取向度高。使用80v01%乙醇-水做后处理剂,采用在后处理剂中先拉伸2倍后浸泡1h的方法制备的纤维性能较初生丝大幅提高,而且存在进一步提高的空间。为了进一步提高纤维的力学性能,摸索最佳处理条件,本论文选择80v01%乙醇-水溶液作为后处理剂,研究了拉伸倍数和浸泡时间对纤维力学性能和结构的影响。结果表明,随着拉伸倍数的增加,纤维的力学性能和取向度增加；相同的浸泡时间下(90分钟),不同拉伸倍数(1-3倍)的后处理纤维的p-折叠构象含量相近。将纤维拉伸3倍后,在后处理剂中浸泡1至120分钟得到纤维的力学性能测试和红外光谱的定量分析表明,随着浸泡时间的增加,后处理纤维的断裂强度和β-折叠构象含量均呈现出开始时快速增长,而后缓慢增长,90分钟后最终趋于恒定的特点。因此本论文暂选的最佳处理条件为,在80v01%乙醇-水溶液中对纤维进行3倍拉伸,然后在其中浸泡90分钟。得到的再生丝素蛋白纤维直径均匀,表面光滑,而且比天然脱胶丝更加强韧。后处理纤维的断裂强度可以达到301 MPa,接近天然脱胶丝。尽管后处理再生丝素蛋白的分子量大大低于天然丝素分子,但是其断裂强度几乎相同,断裂能更是天然脱胶丝的2.4至3.8倍,这表明分子量不是影响再生丝素蛋白纤维力学性能的主要因素,而二级结构才是主要因素。本论文还采用最佳条件对从不同pH(4.8、5.2、5.6、6.0)的再生丝素蛋白水溶液制备的初生丝进行处理。结果表明纺丝液的pH对后处理纤维的结构和力学性能影响非常有限,从不同pH纺丝液制备的初生丝经处理过后具有非常相近的形貌、结构和力学性能,均可以完全媲美脱胶丝。因此,本后处理工艺适用于不同pH值纺丝液制备的初生丝。本论文采用80v01%的乙醇-水溶液作为后处理剂,对再生丝素蛋白干纺纤维进行后处理,得到的纤维性能良好。作为一种后处理剂,乙醇-水溶液无毒环保,具有很好的应用前景。更多还原

【Abstract】 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.更多还原