| Bombyx mori silk fibroin is a kind of animal protein with unique amino acid composition and crystal structure,which is widely used in biomedical,optics and tissue engineering because of its excellent biocompatibility,excellent mechanical properties,non-toxic and biodegradable properties.The use of reconstituted native silk proteins or genetically engineered silks as a raw material,through the bionic green chemical method to obtain high-performance,multi-functional silk-based materials is the basis for the application of silk fibroin.The self-assembling mechanism of silk protein in aqueous solution is of great significance to understand the relationship between its structure and function.At present,there is no uniform and clear understanding of the assembly process and mechanism of silk fibroin in aqueous solution,and it is difficult to monitor the assembly behavior visually.The gold nanoparticles have a surface plasmon resonance(SPR)effect related to their shape,particle size and degree of aggregation.When the gold nanoparticles are aggregated,the plasma coupling between the particles will be triggered to show a visible red to purple or blue change.The application of SPR effect gradually developed into a colorimetric detection method,which is based on gold nanoparticles as indicator,and this method has been applied to colorimetric detection of nucleic acids,small molecules and proteins.Therefore,we present a new idea of using gold nanoparticles as indicator to visibly sense the assembly behavior of silk fibroin in aqueous solution.In the whole sequence of silk fibroin,the highly repeated hexapeptide unit GAGAGS can form an antiparallel beta-folded structure,which in turn forms the crystalline region of silk protein,and its noncrystalline area may formed by the other hexapeptide unit GAGAGY.In recent years,it has become a trend to study the assembly mechanism of silk fibroin by using some representative polypeptide sequences in the silk fibroin sequence as a simplified model.In this paper,the tetradecapeptide(GAGSGAGAGSGAGY,GY-14)and octapeptide(GAGAGAGY,GY-8)were used as the simplified model of the crystalline and amorphous regions of silk fibroin,respectively.The tyrosine at the end of the polypeptide was used as the reducing agent,and gold nanoparticles in-situ reduced of as indicator immobilized on the polypeptide model.The influence of exogenous factors on the assembly behavior of peptide solution was studied by the change of the color and UV absorption peak of the solution.Then,the method of using gold nanoparticles as probe to study the assembly behavior of polypeptide in aqueous solution was studied.The following researches were carried out in this paper: First,the properties of Au NPs dependence of preparation conditions were investigated.The spherical gold nanoparticles with stable dispersion were synthesized by in situ reduction of chloroauric acid using the peptide GY-8 and GY-14 as reductants respectively.The sizes and growth kinetics of Au NPs were influenced by changing the feed ratio,the pH and temperature.The optimal preparation conditions were determined by Ultraviolet-visible spectroscopy(UV-Vis)and transmission electron microscopy(TEM).When the polypeptide GY-8 was used as the reducing agent,the molar ratio of polypeptide to chloroauric acid was 5: 2,solution pH adjusted to 9,the temperature set to 40 °C.When the polypeptide GY-14 was used as the reducing agent,the molar ratio of polypeptide to chloroauric acid was 1:1,the pH adjusted to 7 and the temperature set to 35 °C.Second,the assembly behaviors in aqueous solution of peptide were monitored through in-situ reduced Au NPs as indicator.The self-assembly behavior of the polypeptide GY-14 under the influence of exogenous factors such as pH,metal ion concentration and solvent composition was studied.The assembly method and structure were characterized by UV-Vis,fourier transform infrared spectroscopy(FTIR),TEM and atomic force microscopy(AFM).The results showed that the conformation of the polypeptide GY-14 changed from random coil to ?-sheet in acidic aqueous solution at pH 4,the color of the solution changed from red to purple,and the absorption peak of the UV spectrum shifted red.While the aqueous solution is neutral and alkaline,the polypeptide still retains the random curl,the color of the solution and the position of the UV absorption peak unchanged.The introduction of Ca2+ ion will induce the conformation transformation of the polypeptide.When the ion concentration is 5 mM and 10 mM,the polypeptide will be self-assembled to form ?-sheet structure with a" beads on a string "type of morphology.The solution changed from red to purple and blue,respectively,and UV absorption peak red shifted.The addition of the isopropanol solution resulted in the conversion of the polypeptide from random coil conformation to ?-sheet conformation and the formation of a bead-like assembly with purple flakes in the solution.Afterwards,bead-like assembly precipitated in a short time,UV absorption peak disappeared substantially.In this paper,the assembly behavior of the polypeptide GY-14 in aqueous solution is shown by the change of the color of the solution and the movement of the UV absorption peak with gold nanoparticles as probe,which provides a simple and intuitive new way to study the solution assembly behavior of the polypeptide,mechanism and process of silk fibroin conformation transformation and provide guidance. |
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