Study On The Self-assembly Behavior Of Silk Fibroin In Aqueous Solution By Probe Technique

Silk fibroin,which extracted from natural silk,is a kind of high molecular weight protein with a block structure,it also has unique amino acid composition and conformational structure.because of its biocompatibility,good mechanical properties,and biodegradability,Silk fibroin has great potential applications in medical,biotechnology and tissue engineering Much attention has been paid to artificially preparation of silk-based materials that meet the application requirements from reconstituted silk fibroin.To provide guidance for silk-based material preparation,it is essential to deeply undersand the self-assembly process of silk fibroin in aqueous solutionIt is reported that silk fibroin gradually transforms from a metastable random coil orα-helical conformation to a stableβ-sheet structure during the natural spinning process,which is a self-assembly process.The self-assembly process is regulated by various factors,but the mechanism is not yet clear.Compared to random coils orα-helices conformations,molecules in silk fibroin are organized and have smaller molecular distances withβ-sheet conformation.With changes in conformation,the microenvironment of silk fibroin is also changing。A variety of methods have been developed to investigate the assembling process,however,most of them have some drawbacks such as requiring complicated sample preparation,expensive equipment or certain analytical technique,which make the cost of research has been greatly increased.To develop easily prepared,inexpensive and simple monitoring methods for assaying assembly behavior of silk fibroin is still desired.The SPR effect of gold nanoparticles affords them widely applicable to colorimetric detection of heavy metal ions,peptides,proteins,and cancer cells.Fluorescent probes have been widely used due to its advantages of high sensitivity,good specificity,and non-destructive examination.It is explored to use probes technique for monitoring the conformation changes in silk fibroin in this article.The following studies are conducted:（1）The assembly behaviors in aqueous solution of silk fibroin were monitored through in-situ Au NPs as indicator.Firstly,the optimal reaction conditions for the in situ preparation of gold nanoparticles by silk fibroin were explored by UV-Vis spectroscopy:Silk fibroin concentration is 2 mg·mL^-1,Chloroauric acid concentration is 2 mM,reaction temperature is 40℃.Then the effect of pH on the self-assembly of silk fibroin was studied using gold nanoparticle probes.The results show that the UV absorption peak red shifted under acid condition,accompany with a color change of the solution from red to purple.However,as shown in TEM images,the red shift of absorption peak is induced by growing up of Au NPs rather than the interparticle distance changing.So the prepared Au NPs are not stable under acid condition.Moreover,it is further found that the interaction of gold nanoparticles with silk fibroin affects the assembly process of silk fibroin through circular dichroism spectroscopy characterization.Therefore,gold nanoparticles are not suitable as probes.（2）The self-assembly behaviors of silk fibroin under the influence of pH and metal ions are studied respectively by using Th T and ANS as fluorescent probes.The system is characterized by fluorescence spectroscopy,infrared spectroscopy,transmission electron microscopy and atomic force microscopy.The results show that the fluorescence intensity gradually increases with the decrease of pH,indicating that theβ-sheet conformation content of silk fibroin gradually increases in the system.Other characterization methods also verify the experimental results.Metal ions can promote the formation ofβ-sheet conformation within a certain range(Ca²⁺<0.5 mg·g^-1,K⁺<1 mg·g^-1).beyond this concentration range,the fluorescence spectrum intensity of the system gradually decrease with increasing ion concentration,indicating that excessively high concentration of ions would inhibit the conformational change.The characterization of TEM and AFM also proved that the ion and protein interactions will affect the assembly process when the metal ion concentration is too high.Under different concentrations of ions,silk fibroin shows different assembly forms.When the concentration is low,fibroin forms filamentous fibers,and when the concentration is high,fibroin forms spherical fibrils...