Applied Basic Research On Assembled Multilayers Of Polyelectrolytes: Fabrication And HRP Assembly

Layer by layer (LBL) assembly method with the advantage of versatility, simplicity and easy operation was widely applied, because the thickness, structure and function of films obtained were controllable. The study of fabricating the films with tailored bioactivity or other function by using LBL technique was very active. Thus applied basic research of LBL has received considerable interest.In this paper, the fabricating of PAH/PSS and PDDA/PSS multilayer and the driving force and pattern of HRP film assembled on the surface of PAH/PSS multilayer or negative ionized PET were studied. Influence of assembling condition on the growth of PAH/PSS and PDDA/PSS multilayer was investigated comparatively by using UV-Vis and AFM techniques and the intrinsic reason of difference of multilayer growth regime was revealed. After systematically investigating the effect of experimental condition on the bio-catalytic activity of HRP assembly film, the driving force and pattern of HRP assembled on the charged surface was discussed. Furthermore, the application of HRP enzyme film assembled on the negative ionized PET to spectrophotometric analysis was studied by using micro cuvette as reactor.The growth regime of multilayer related to molecular structures of polyelectrolyte. PAH/PSS multilayer composed of flexible PAH was linear growth and PDDA/PSS multilayer of rigid PDDA was exponential growth. The AFM images distinctly showed the difference of the surface structure of PAH/PSS and PDDA/PSS multilayers. The results of researching on the deposition time, concentration of polyelectrolyte and NaCl in assembly solution indicated that the experimental condition can change the growth speed but the growth regime of multilayer.Treating assembly amount of multilayer using geometric progression, the mathematics equation describing three type of growth regime was derived. The parameters, which have certain physic meanings, were capable of explaining the intrinsic difference of growth regimes. This provided a new route understanding the growth regime and structure of polyelectrolyte multilayer.The pattern and driving force of HRP assembled on the surface of PAH/PSS multilayer were studied. When the outlayer of PAH/PSS multilayer was PSS, HRP deposited on the surface of multilayer and reached balance in Ih. When the outlayer of PAH/PSS multilayer was PAH, HRP embedded in the PAH layer and reached balance after 16h. The assembly amount of HRP on PAH layer was 2-4 times large than on the PSS layer. The effect of experimental condition on the activity of HRP assembly film was investigated in detail. The results indicated that driving force of IIRP assembled on the multilayer was the electrostatic interaction between the ionized group in HRP molecular and on the surface of multilayer.The LBL premixing multilayer of HRP was self-assembled by using the mixing solution of PSS and HRP as polyanion and PDDA as polycation. The activity of HRP on premixing film increased with increasing HRP concentration in premixing solution. The lower activity of premixing film obtained in the pH of premixing solution close to the pi of HRP indicated that HRP was premixing assembled in film drove by various interactions. The banded structure of premixing film observed by AFM was different apparently with the film assembled using other method.Application of HRP film assembled on the surface of negative ionized PET in spectrophotometric analysis was researched. The color reaction of H2O2 and 4- amino antipyrine catalyzed by HRP film finished in 5min. The low Michaelis constant indicated the strong affinity between'H2O2 and HRP on film. The relative standard deviation (RSD) of sample measuring was 1.9% and the recoveries of H2O2 were 96.5%~ 101.1%. After 150 days, the relative activity still remained above 80%. The activity stability of HRP in storing at 4°C was very good. HRP/PET enzyme film was able to apply in the spectrophotometric determination of H2O2 effectively.