Synthesis Of Polymer/Protein Core-Shell Nanoparticles And Their Application In Glucose Biosensors

With a title of "Synthesis of Polymer/Protein Core-Shell Nanoparticles and Their Application in Glucose Biosensors", the dissertation focuses on the synthesis of core-shell nanoparticles with a protein shell by copper mediated graft copolymerization; the core-shell structure of these particles are characterized in detail by a combination of laser light scattering (LLS), transmission electronic microscopy (TEM), Zeta potential and X-ray photoelectron spectra (XPS) techniques; the interaction and adsorption of enzyme on these particles with a protein shell are studied by using a quartz crystal microbalance with dissipation (QCM-D) and LLS; the biosensor is constructed by immobilizing enzyme on these particles with a protein shell, and the analytical performance of the biosensor is investigated by cyclic voltammetry and chronoamperometry. The main results are as follows:First, well-defined core-shell poly(methyl methacrylate)-bovine serum albumin (PMMA-BSA) particles have been prepared by direct graft copolymerization of methyl methacrylate (MMA) on bovine serum albumin (BSA) with a trace amount of copper ions. The core-shell structure of PMMA-BSA particles was confirmed by the TEM analysis. The pH dependence of the Zeta potential of PMMA-BSA particles and pure BSA is similar, indicating BSA is coated on the PMMA-BSA particles as a shell. The XPS analysis reveals that the XPS surveys of the PMMA-BSA particles and pure BSA are similar, further demonstrating that the surface of the PMMA-BSA particles is covered by BSA. Effects of the reaction temperature, the mass ratio of BSA to MMA, and the copper ions concentration on the copolymerization have been studied. The principle of copolymerization was investigated by characterizing the structure of PMMA homopolymer isolated by the Soxhlet extraction and PMMA grafted on BSA obtained by hydrolyzed with acid. The results reveal that copper mediated graft copolymerization of MMA on BSA is free radical polymerization, similar to an emulsion polymerization. The PMMA-BSA core-shell particles are formed by the self-assembly of those amphiphilic PMMA-BSA grafted copolymer chains, initiated by radical produced from copper complex with BSA under heating. The average particle size can be well controlled in the range 60-110 nm with a variation of the initial mass ratio of MMA to BSA. Such formed core-shell particles have a biocompatible BSA shell. Further, we studied the immobilization of glucose oxidase (GOx) on such prepared PMMA-BSA particles. The interaction and adsorption of GOx on PMMA-BSA particles were investigated by a combination of QCM-D and LLS measurements. The results show that the adsorption of GOx on PMMA-BSA particles is driving by electrostatic interaction. Both PMMA-BSA and GOx are positively or negatively charged when pH<4.2 (the pI of GOx) or>5.1 (the pI of PMMA-BSA) so that the electrostatic repulsion leads to no adsorption of GOx on PMMA-BSA in these two ranges; the adsorption occurs at pH between these two pI values, at which they are oppositely charged. The QCM-D studies reveal that the adsorption kinetics contains two processes:the quick adsorption is due to electrostatic interaction; the second process is attributed to the continued adsorption of GOx on the new points produced by the conformation change of adsorbed GOx. The enzymatic activity was determined by UV/vis measurements. The result shows that the adsorbed GOx can retain more than 80% activity in comparison with free enzyme. Thermal stability studies reveal that the adsorbed GOx only losses 28% of its activity in comparison with a 64% activity loss of free GOx when they are incubated at 50℃for 35 h.On the basis of these studies, we have constructed an amperometric glucose biosensor by immobilizing GOx on PMMA-BSA particles. The performance of prepared biosensors was tested by cyclic voltammetry and chronoamperometry. The testing results demonstrate the biosensor has a good performance in the oxidation of glucose. The cyclic voltammetry measurement reveals that the biosensor involves a typical diffusion-controlled electrochemical reaction and has a linear response in the glucose concentration range 0.2-9.1 mM with a sensitivity of 44.1μA mM-1 cm-2 and a short response time of 6 s under an working potential of 0.4 V. It is worth noting that the biosensor keeps a relatively high response current (25.8μA mM-1 cm-2) when a working potential 0.25 V is used; namely, it can be used to eliminate the interference by working at low potential. The reproducibility was tested by using six electrodes prepared in the same way and an identical glucose concentration (1 mM). The relative standard deviation is 4.9%, showing a good reproducibility of the biosensor preparation. The durability test demonstrated that there is nearly no change of the response current even after 50 successive scans in a glucose solution of 1 mM. More important, such prepared biosensors are thermally stable at the room temperature; namely, the response current of the biosensor only decreases～4% after they were stored for 23 days. After 30 days, the biosensor still retains～82% of its original response. The excellent operational and storage stability make these biosensors potentially useful in biomedical application.