| Amphiphilic block polymers including nature and synthetic polymers have attracted much attention because of their widespread applications and relatively complex behaviors. Because of many good properties such as low cost, stability, safety and good biocompatible of nature amphiphilic polymers (e.g. casein, gelatin, and chitosan), they have been shown to exhibit properties beneficial to food, cosmetic and medical science and will have significant implications in advances of future generations of materials. It is well-known that the environment conditioning can significantly alter the behavior and overall performance of biopolymers, which provides more choices for the wide use in practice. Therefore, in our work, the structure-property relationship of casein micelles modulated by the environment conditioning has been deeply studied. Furthermore, the interaction between casein micelles and gold nanoparticles, as well as the synthesis of gold nanoparticles in casein micelles has also been studied. The results will broaden the application range of the casein in food, cosmetic and medicine domains. The following are some main results from our work:1. The association behavior of the casein over a broad pH range has been firstly investigated by fluorescent technique together with CD, DLS and turbidity measurements. Casein molecules can self-assemble into casein micelles in the pH range 2.0 to 3.0, and 5.5 to 12.0. The hydrophobic interaction, hydrogen bond and electrostatic action are main interactions in the formation of casein micelle. The casein micelle has the most compact structure at pH 5.5, when the casein micelle has almost zero charge. The structure of the casein micelle becomes looser with the increase of pH because of the stronger electrostatic repulsive interaction. The compact extent of casein micelle structure plays an important role in the binding of pyrene molecules to casein micelles. The rigid configuration of pyrene molecules limits the affinity of pyrene molecules to the hydrophobic domain of casein micelles with more compact structure. ANS has high affinity with casein micelle only at acidic condition and the affinity decrease sharply with the increase of pH. Therefore, apart from the hydrophobic interaction, the electrostatic interaction between ANS and casein micelles plays an important role in the binding of ANS to casein micelles. The sheet structure transfer to helix and turn structure during the self-assembling process of casein, and more helix structure are formed at acid condition.2. The interactions between the cationic surfactant dodecyltrimethylammonium bromide (DTAB) and 2.0 mg/ml casein were investigated using isothermal titration calorimetry (ITC), turbidity, dynamic light scattering (DLS), and fluorescence spectra measurements. At DTAB concentration lower than the c1 (1.38 mM), the cationic headgroups of surfactant individually bind to the negative charged amino acid sites on the casein chains due to electrostatic attraction, which results in an increase of△Hobs. At the same time, the structure of casein micelles becomes more compact due to the decrease in the net negative charge of the casein micelle shell, and hence a decrease of the casein micelle's hydrodynamic radius. When the DTAB concentration exceeds c1, the casein bound surfactant aggregates result in the formation of large insoluble casein/surfactant complexes because of the markedly electrical neutralization of negative charge of casein micelles by cationic DTAB. This leads to the sharp increase of the turbidity of the system beyond c1. Therefore, the turbidity increases sharply and then reaches a maximum value with the addition of DTAB. Beyond c′, the net positive charges on the complexes owing to the binding by more cationic surfactant molecules lead to a redissolution of the complexes, corresponding to the formation of the new casein/DTAB complexes. At DTAB concentration of 18 mM (c2), all the caseins are saturated by DTAB aggregates and free DTAB micelles appear in solution. In excess of salt, where the electrostatic attractive force between casein and surfactant is considerably screened, and the electrostatic repulsion between the surfactant headgroups is also shielded by the addition of salt, which favors the formation of free micelles of DTAB.3. The influence of the typical anionic surfactant sodium dodecyl sulfate (SDS) on the properties of casein micelles was investigated using fluorescence spectra, isothermal titration calorimetry, CD, DLS and TEM techniques. The hydrophobic interaction between SDS and casein micelles leads to important changes in physicochemical parameters of casein micelles. At SDS concentration lower than critical aggregation concentration (c1), SDS monomers bind to the hydrophobic domain of the casein micelles by hydrophobic interaction. When SDS concentration reaches c1, the micelle-like SDS aggregate are formed in casein chain. When SDS concentration reaches the critical micelle concentration (c2), free SDS micelles coexist with casein/SDS complexes in the system. The intrinsic fluorescence results show that the addition of SDS hampers the energy transfer between Trp and Tyr residues after SDS binding to the hydrophobic domain of casein micelles. The extrinsic probe (pyrene and ANS) fluorescence results show that the bound SDS molecules limit the formation of pyrene excimer, but don't affect the microenvironment polarity around pyrene molecules. In addition, the addition of SDS decreases the affinity of ANS to casein micelles obviously because of the electrostatic repulsive interaction between ANS and SDS. CD measurements show that the addition of SDS leads to the variation of the second structure of casein molecules. TEM imagines and DLS confirm the formation of casein-SDS mixed micelles. Thus, it seems that the presence of the anionic surfactant can be a tool to control the size and properties of casein aggregates in solution.4. We report the spontaneous, in-situ synthesis of gold nanoparticles within casein micelles. Simple and convenient methods were employed that resulted in gold particle formation as asserted by UV-vis spectroscopy and transmission electron microscopy (TEM), selected area-electron diffraction (SAED) and XRD measurements. Au colloids with various morphologies (e.g., spherical nanoparticles, triangular, hexagonal plates and decahedron) are formed through auto reduction of HAuCl4 in casein aqueous solution at room temperature without any additional chemical. The casein molecules provided the dual function of Au(III) reduction and directing the anisotropic growth of Au (0) into plate-like structures. XRD and SAED showed that the nanoplates were oriented with {111} planes as their basal planes. There is no additional template agent in the synthesis process, which makes the synthetic procedures and the related treating processes very simple. The present synthetic route is fast and the size of the resultant nanosheets is very large, and it is favorable to produce gold nanosheets in large scale. The percentage of the gold nanotriangles and decahedron can be easily varied by merely adjusting the raio of Au/casein. These are most likely determined in terms of a competition between metal ion reduction activity and on the surface of particles and/or among particle aggregates and the colloidal stabilization modulated by the casein molecules. In the presence of Au seed, large portion of spherical gold particles, and only a small portion of triangle gold particles are formed. The effect of pH on the formation of gold nanoparticles suggests that the protonated amino plays an important role in the reduditon of Au(III).5. For the first time, the interaction of casein micelles (CMs) with gold nanoparticles (GNPs) was studied using UV-visible spectroscopy, TEM, fluorescence spectroscopy, andζ-potential measurements, respectively. The red shift in the position of the plasmon absorption band is produced by a perturbation in the dielectric constant around GNPs due to their adsorption on CMs. No significant broadening of the spectrum is observed after the adsorption, indicating that the GNPs do not experience aggregation into larger nanoparticles upon the adsorption on CMs. TEM results show that the gold particles also exhibit similar particle diameter, but appear clustered on the micrographs rather than being evenly dispersed in the presence of CMs. In addition, the typical GNP cluster takes on spherical with a diameter of about 250 nm. Fluorescence studies further indicate the GNPs are located on the CM surfaces, and the CM structure is retained after the adsorption of GNPs. UV-visible spectroscopy also shows that the GNP-CM conjugates display good stability toward salt concentration and pH. The combination of these measurements suggests that GNPs bind to CM surfaces via complexation with the carboxylate, amine or S groups on CM surfaces and hydrophobic interaction, but not by electrostatic action. The proximity of GNPs on casein micelle surface to Trp residues differs much at different pH, and in the presence of different salt.
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