Synthesis Of PVDAT Nanoparticles With Controllable Particle Size And Studies On Enzyme Adsorptions In Aqueous Phase

Functional polymer nanoparticles have the characteristics of large specific surface area,multiple active functional groups,high surface activation energy,and excellent adsorption performance.They have been widely used in the fields of analysis and detection,enzyme immobilization,biomedicine and protein molecularly imprinted polymers.At present,most polymers for protein adsorption cannot form stable physical interactions with proteins in aqueous phase wherea they likely destroy the natural conformation of the biological macromolecules,leading to the loss of enzyme activity,thus limiting their applications.To tackle these fundamental and technical challenges,the work presented in this thesis employed the semi-continuous aqueous precipitation polymerization method to prepare polymer nanoparticles with controllable particle size,and studied their adsorption of enzymes in aqueous phase.In this study,2-vinyl-4,6-diamino-1,3,5-triazine(VDAT)was selected as the functional monomer based on a hypothesis that the diamino triazine functional groups hold potentials to form stable hydrogen bonds with target proteins in aqueous phase due to the monomer's unique chemical composition and spatial structure.VDAT polymer(PVDAT)nanoparticles(NPVDAT)with a particle size of 150 nm and good monodispersity were successfully synthesized by semi-continuous aqueous precipitation polymerization.After purification by water dialysis,the stable particle dispersion was maintained and the zeta potential increased from +38.89 m V to +51.97 m V,indicating that the system had become more stable.In addition,the polymerization method was proved to be feasible of controlling the size of NPVDAT with a minimum particle size of 50 nm,which laid the foundation for fabrication of multifunctional core-shell structure polymer particles.The particle size of NPVDAT was controlled by simply changing the monomer loading and the relationship between the polymer particle size and monomer loading was obtained.The mechanism of NPVDAT formation had been discussed.Briefly,VDAT oligomers formed at the early polymerization stage precipitated from the aqueous phase and aggregated to form nuclei which then grew by adsorbing more and more VDAT oligomers/polymers.The particle growth continued as more monomer was fed into the reactor.As such,the particle size was readily controllable by adjusting the total monomer loading.The successful synthesis of NPVDAT enabled further studies on the effects of particle size on protein adsorption.Furthermore,the synthetic method developed in this study provides a novel and viable approach for preparing functional nanoparticles with controllable particle size.Through NPVDAT's adsorption study on horseradish peroxidase(HRP)and trypsin(TRY),it was confirmed that stable hydrogen bonding formed between NPVDAT and the protein,leading to strong and stable physical adsorption,and the hydrogen bonding effect was greater than the electrostatic effect in the aqueous phase.In the adsorption study of HRP,the higher the p H,the larger the adsorption capacity,owning to the synergistic effect of hydrogen bonding and electrostatic interaction.Ionic strength affected dispersion stability of the nanoparticles,and the adsorption efficiency decreased with the increase of ionic strength.The three adsorption isotherm models of Langmuir,Freundlich and Temkin were used to fit the adsorption data of HRP.It was found that the Langmuir model was the most suitable for explaining the adsorption mechanism.This suggests a monolayer adsorption of HRP by NPVDAT with a maximum adsorption capacity of 13.80 mg/g.Adsorption kinetic studies indicated that the adsorption equilibrium was reached in 35 minutes.The effect of NPVDAT particle size on HRP adsorption showed that the particles with smaller size had a larger adsorption capacity,which fully reflects the superiority of the nano-materials as adsorbents.Finally,the ELISA analysis indicated that up to 74.99% of the HRP adsorbed by NPVDAT were biocatalytic active.The adsorption study of TRY by NPVDAT also demonstrated the dominant role of hydrogen bonding.The adsorption mechanism was also in line with Langmuir monolayer adsorption,and the maximum adsorption capacity was 269 mg/g.By studying the influence of the particle size of NPVDAT on the adsorption of TRY,on the one hand,the smaller the particle size,the larger the adsorption capacity.On the other hand,when the NPVDAT was too large,the surface curvature would be small,and the TRY macromolecules would spread out on the particle surface due to the strong physical interactions,resulting in the protein conformational changes and hence,enzyme denaturing.The activity of TRY was quantified by the reaction of TRY hydrolyzing N-?-benzoyl-L-arginine ethyl ester hydrochloride.The results showed that in a wide range of enzyme concentrations,the adsorbed TRY maintained about 70% of the activity.The above adsorption study results demonstrated that the adsorption at NPVDAT surface did not significantly change the protein conformation,which laid a practical foundation for the next step in exploring the preparation of biosensors and protein molecularly imprinted polymers.