【作者】 黄继伟；

【导师】 姚穆； 左保齐；

【作者基本信息】 苏州大学 ， 纺织材料与纺织品设计， 2022， 博士

【摘要】 甲酸(FormicAcid,FA)与氯化钙(CaCl2)配合形成的溶剂体系,可将脱胶蚕丝(Silk Fibroin Fiber,SFF)溶解至“原纤级”。本文发现SFF在FA溶剂体系中经历了“溶胀→分纤→原纤级溶解”的过程,针对此过程的研究将有利于揭示SFF在FA溶剂体系中发生“原纤级”溶解的机理,可为丝素蛋白基再生材料的制备、性能调控及应用提供技术手段、基础数据和理论支撑。另一方面,SFF作为一种具有各向异性的蛋白质纤维,其在横向的溶胀,则在纵向往往表现出收缩的现象,针对这一溶胀收缩现象及相关结构与性能变化的研究,是认识和揭示蛋白质纤维层级结构及构效关系的常用手段。蜘蛛丝在高湿空气或水中的超收缩、蚕丝在中性盐(如CaCl2、溴化锂(LiBr)、硝酸钙(Ca2(NO)3)等)水溶液中发生的溶胀收缩等均是相关领域研究的典型实例。本文系统探讨SFF在FA溶剂体系中发生的剧烈溶胀与显著收缩,直至溶解的行为,并以此对SFF的结构、性能,及构效关系进行深入讨论。首先,本文研究了不同蚕丝(包括:脱胶桑蚕丝(Mulberry Silk Fibroin Fiber,MSFF)、脱胶柞蚕丝(Tussah Silk Fibroin Fiber,TSFF)和脱胶木薯蚕丝(Cassava Silk Fibroin Fiber,CSFF))在FA中发生溶胀收缩的行为及关键影响因素。结果发现,MSFF在FA中可发生显著的溶胀收缩,FA浓度是影响MSFF溶胀收缩的关键因素,FA浓度越高,MSFF的溶胀收缩越显著,在98 wt.%FA中MSFF的收缩率可达50%以上,而TSFF和CSFF在FA中的溶胀收缩却十分有限,两者的最大收缩率不足20%。分析认为,FA分子与丝素肽链分子中极性氨基酸侧基(包括甘氨酸)间的交互作用是促使MSFF发生显著溶胀收缩的初始动力,基于此动力,FA进入MSFF内部,促使了 MSFF横向的溶胀和纵向的收缩。而TSFF和CSFF中极性氨基酸侧基的含量不及MSFF,且拥有高度的聚丙氨酸([Ala]n)结晶结构,进入TSFF和CSFF内部的FA量少,使得两者的溶胀收缩有限。接着,本文对各种SFF经不同浓度FA溶胀收缩后的结构与性能进行了表征,以进一步探究SFF在FA中发生溶胀收缩的机制。结果发现,SFF经不同浓度FA溶胀收缩后,其取向度随着所浸泡FA浓度的提高而降低,MSFF取向度的变化幅度显著大于TSFF和CSFF,三者的结晶度变化并不显著;同时,MSFF经较高浓度(≥ 92wt.%)FA浸泡后,其原纤间连接带的均匀性变差,不规则孔洞增多,而MSFF经较低浓度(≤90wt.%)FA浸泡后及TSFF和CSFF经不同浓度FA浸泡后的原纤间连接带的均匀性却有所改善。分析认为,当FA进入SFF内部的量较少时(包括MSFF经较低浓度FA浸泡、TSFF和CSFF经各种浓度FA浸泡),FA分子未在SFF内形成显著集聚,未能改变原纤的绞状形式,取向度变化较小,而FA分子与SFF肽链分子中极性氨基酸侧基的交互作用,又使得SFF内原纤间连接带的均匀性被改善。当FA进入SFF内部的量较多时,可扰乱SFF内原纤的纵向排列,呈绞状形式存在的原纤出现解绞,使得SFF在微观上表现为取向度降低,在宏观上表现出显著的绕轴旋转、横向溶胀和纵向收缩现象。从力学性能角度分析,SFF经FA浸泡后的取向度下降和原纤间连接带的扰动决定了其断裂伸长率提高和断裂强度下降。然后,基于MSFF在FA中的显著溶胀收缩行为,进一步研究了受限收缩和收缩后再拉伸对MSFF结构与性能变化的影响。结果发现,在高浓度(98 wt.%)FA中受限收缩MSFF的取向度较同浓度下自由收缩的大,结晶度变化不显著,但其原纤间连接带的均匀性却进一步变差;而高浓度FA中收缩后再拉伸的MSFF,其结晶度变化同样不显著,取向度和原纤间连接带的均匀性则随着拉伸率的提高而增加,在拉伸至原长的120%时,其原纤间连接带的均匀性被还原至未经FA浸泡时的状态。分析认为,MSFF在高浓度FA中受限收缩时,原纤间存在滑移和错位,使原纤间起到连接作用的分子链被拉伸,而收缩后再拉伸又可使原纤伸直,使连接原纤的分子链拉伸程度被进一步提高,并导致了受限收缩和收缩后再拉伸MSFF相对于其自由收缩的初始模量增加、断裂伸长率下降和断裂强度提高。再者,针对TSFF和CSFF在FA中的溶胀收缩不显著,通过在高浓度FA中添加CaCl2的方式,使TSFF和CSFF发生进一步的溶胀收缩。基于此手段,研究了 TSFF和CSFF经不同浓度CaCl2-FA溶胀收缩后的结构与性能变化。结果发现,TSFF和CSFF可在CaC12-FA中发生显著的溶胀收缩,且其收缩后的取向度显著下降,结晶度变化不显著,原纤间连接带的均匀性被严重破坏;在一定的CaCl2浓度范围(<4 wt.%)内,TSFF和CSFF的断裂伸长率提高,超过此范围后,TSFF和CSFF的断裂伸长率则开始下降。分析认为,FA中的CaCl2可进一步使TSFF和CSFF原纤间连接带内分子链发生卷曲,当FA中CaCl2浓度较高时,TSFF和CSFF的原纤间连接带内的部分分子链已发生水解断裂,导致了其断裂强度和断裂伸长率的急剧下降。最后,本文研究了 FA溶剂体系溶解MSFF的机理,发现CaCl2-FA溶剂体系中CaCl2含量与FA浓度的配置是影响MSFF溶解的关键因素。随着FA浓度的降低,MSFF的溶解度急剧下降,但通过增加CaCl2浓度的方式可提高MSFF的溶解度;在一定的CaCl2浓度范围(<10wt.%)内,MSFF溶解物的结构并无显著的差异,超过此范围后,所得MSFF溶解物的分子量降低、Silk I结晶形式的含量增加。结合CaCl2具有破坏/限制丝素蛋白结晶的作用分析和HCl辅助FA溶解MSFF导致其分子量降低的分析,认为在FA分子聚集导致的溶胀作用、Ca2+离子与丝素肽链分子间的配位键作用、H+和Cl-离子的催化水解作用等的共同影响下,SFF发生了溶胀、分纤和原纤级溶解,使其再生丝素蛋白材料表现出了显著的原纤化特征。综上,在FA溶剂体系中SFF经历了“溶胀→分纤→原纤级溶解”过程,在此过程中,FA能使SFF的原纤间连接带发生显著的形变,而CaCl2的加入又使这种形变加剧,当CaCl2浓度较高时,使原纤间连接带内起连接作用的分子链水解和断裂,最终导致了 SFF原纤被溶解至溶剂中。更多还原

【Abstract】 The solvent system formed by formic acid(FA)and calcium chloride(CaCl2)could dissolve degummed silk fiber(Silk Fibroin Fiber,SFF)to "fibril level".In this work,it was discovered that SFF undergoes a process of "swelling→fibrils splitting→fibril-level dissolution" in the FA solvent system.The study focusing on this process would help to reveal the fibril-level dissolution mechanism of SFF in the FA solvent system,and provide the technical means,basic data,and theoretical support for the preparation,performance tuning,and application of silk fibroin-based regeneration materials.On the other hand,SFF,as an anisotropic protein fiber,tends to contract in the longitudinal direction when it swells in the transverse direction.It is a popular way to understand and reveal the hierarchical structure and structure-function relationship of protein fiber.The supercontraction of spider silk in high humidity air or water and the swelling contraction of silk in neutral salt(such as CaCl2,lithium bromide(LiBr),calcium nitrate(Ca2(NO)3)aqueous solution were typical research cases in related fields.In this work,the severe swelling,supercontraction,and dissolution behavior of silk fiber in FA solvent system were systematically investigated and analyzed,to deeply discuss the structure,properties,and structure-function relationship of silk fiber.Firstly,the behavior and key factors affecting the swelling contraction of different silks(including degummed mulberry silk fiber(Mulberry Silk Fibroin Fiber,MSFF),degummed tussah silk fiber(Tussah Silk Fibroin Fiber,TSFF),and degummed cassava silk fiber(Cassava Silk Fibroin Fiber,CSFF))in FA were investigated.The findings revealed that MSFF could swell and contract significantly in FA,and the concentration of FA was the key factor affecting the swelling contraction of MSFF,the higher the FA concentration,the more significant the contraction of MSFF,and the contraction rate of MSFF in 98 wt.%FA could reach more than over 50%,However,the swelling and contraction of TSFF and CSFF in FA was very limited,with maximum contraction rate of less than 20%.The analysis suggests that the interaction between FA molecules and polar amino acid side groups(including glycine)in silk peptide chain molecules was the initial driving force to promote the significant contraction of MSFF.Based on this driving force,FA could enter the interior of MSFF,which promotes the horizontal swelling and vertical contraction of MSFF.However,the content of polar amino acid side groups in TSFF and CSFF was lower than that of MSFF,TSFF and CSFF had a high content of polyalanine([Ala]n)crystal structure,which resulted in less FA penetration,as a result,the swelling contraction of TSFF and CSFF was limited.Subsequently,the structure and properties of various SFFs treated with different concentrations of FA were characterized,to further explore the mechanism of swelling contraction of SFF in FA.The results showed that the orientation degree of SFF decreased with the increase of FA concentration,and the change range of orientation degree of MSFF was significantly higher than that of TSFF and CSFF,all the crystallinities do not change significantly.Meanwhile the uniformity of fibrils bonding ties became worse and irregular holes increased for MSFF treated with higher concentration(≥ 92wt.%)FA.However,the uniformity of fibrils bonding ties was improved for MSFF treated with lower concentration(≤90wt.%)FA and TSFF,CSFF treated with different concentrations of FA.It was considered that when the amount of FA into the SFF was less(including MSFF treated with low concentration FA,TSFF and CSFF treated with various concentrations of FA),FA molecules do not form significant agglomeration in the SFF,fail to change the twisted form of fibrils,and the change of orientation was small.However,the interaction between FA molecules and the side groups of polar amino acids in SFF peptide chain molecules improves the uniformity of fibrils bonding ties in SFF.On the other hand,when the amount of FA into the SFF was larger,FA could disturb the longitudinal arrangement of fibrils in the SFF,and the fibrils in the form of twist appear to be untwisted,which reduces the orientation degree of SFF at microscopic level and shows significant phenomena of axial rotation,transverse swelling and longitudinal contraction at macroscopic level.From the point of view of mechanical properties,the decreased orientation degree of SFF soaked with FA and the disturbance of fibril bonding ties determine the increase of elongation at break and the decrease of breaking strength of SFF.Then,based on the significant swelling contraction behavior of MSFF in FA,the effects of restricted contraction or re-stretching after contraction on the changes in structure and properties of MSFF were further studied.The results showed that the orientation degree of the restricted contraction MSFF in high concentration(98 wt.%)FA was higher than that in free contraction at the same concentration of FA,and the change of crystallinities was not significant,but the uniformity of fibril bonding ties became further worse,The crystallinity of MSFF re-stretched after the contraction in high concentration FA also did not change significantly,and the orientation and the uniformity of fibrils bonding ties increased with the increase of re-stretching rate.When the fiber was stretched to 120%of the original length,the uniformity of fibrils bonding ties was restored to the state of MSFF without FA treatment.It was considered that when MSFF was subject to the restricted contraction in high concentration FA,there was slip and dislocation between the fibrils,resulting in the molecular chains connecting the fibrils to be stretched.The re-stretching after contraction could straighten the fibril,which can further improve the stretching degree of the molecular chain connecting the fibrils in MSFF,and also lead to the increase of the initial modulus,the decrease of elongation at break and the increase of breaking strength of MSFF relative to its free contraction.In addition,the swelling contraction of TSFF and CSFF in FA was not significant,which could be significantly enhanced by adding CaCl2 to high concentrations of FA.Based on this method,the changes in structure and properties of TSFF and CSFF after swelling contraction with different concentrations of CaCl2-FA were investigated.The results show that TSFF and CSFF could contract significantly in CaCl2FA,and the orientation degree after contracting decreased significantly,the change of crystallinity was not significant,and the uniformity of the bonding ties between fibrils was seriously damaged.Within a certain CaCl2 concentration range(<4 wt.%),the elongation at break of TSFF and CSFF increased.Beyond this range,the elongation at break of TSFF and CSFF began to decrease.The analysis shows that CaCl2 in FA could further curl the molecular chain in the bonding ties between fibrils of TSFF and CSFF.When the concentration of CaCl2 in FA is high,some molecular chains in the bonding ties between fibrils of TSFF and CSFF had broken,resulting in a sharp decrease of their strength and elongation at break.Finally,the paper explored the dissolution mechanism of MSFF in FA solvent system.It was found that the content of CaCl2 and the concentration of FA were key factors affecting the dissolution of MSFF.The solubility of MSFF decreased sharply with the decrease of FA concentration;When the concentration of FA was low,the solubility of MSFF could be improved by increasing the concentration of CaCl2;In a certain concentration range of CaCl2(<10wt.%),there was no significant difference between the structure of MSFF solutes.Beyond the range,the molecular weight of the resulting MSFF solutes decreased and the content of Silk I crystal line form increased.Combined with the analysis of CaCl2 in destroying/limiting crystallization of silk protein and the dissolution of MSFF by FA with the aid of HCl resulting in the decrease of its molecular weight,it was regarded that SFF undertakes swelling,fiber splitting,and fibrils-level dissolution due to the swelling role caused by FA molecular aggregation,the coordination bond between Ca2+and silk fibroin molecules,and the catalytic hydrolysis of H+and Cl-,which endows the regenerated silk fibroin materials with fibrillation characteristics.In summary,the "swelling→fiber splitting→fibrils level dissolution" of SFF in FA solvent system mainly occurs in the bonding ties between fibrils of SFF.FA could significantly deform the bonding ties between fibrils of SFF,and the addition of CaCl2 aggravates this deformation.When the concentration of CaCl2 was high,the molecular chain connecting the fibril would be hydrolyzed and broken,resulting in the dissolution of SFF in the form of fibrils.更多还原