Protein Multilayers

In many cases, one can consider proteins as hard monodisperse nanoparticles with dimensions of 2-10 nm. Taking protein solution at pH well apart from its isoelectric point, one can provide 10-60 elemental charges for a protein globule due to ionization of its carboxyl or amine groups [77]. Multilayer films that contain ordered layers of protein species were assembled by means of alternate electrostatic adsorption mostly with positively charged PEI, PAH, PDDA, and chitosan or with negatively charged PSS, DNA, and heparin [77-96]. The pH of the protein solutions was set apart from the isoelectric point so that proteins were sufficiently charged under the experimental conditions. The assembly of 20 different proteins was successfully achieved (including cytochrome, carbonic anhydrase, myoglobin, hemoglobin, bacteriorhodopsin, pepsin, peroxidase, alcohol dehydroge-nase, glucoamylase, glucose oxidase, immunoglobulin, cata-lase, and urease) (Fig. 5) [79]. The mass increment at each

Figure 5. SEM image of cross-section of glucose oxidase/PEI multilayer (24 bilayers, thickness 270 nm) on quartz support.

step was quite reproducible. Proteins immobilized in multilayers with strong polyions such as PSS, PEI, and PDDA were insoluble in buffer for a pH range between 3 and 10. The assembled proteins are in most cases not denaturated [77-78, 82, 87-88]. Moreover, the layer-by-layer immobilization with linear or branched polyions enhanced the enzymatic stability [95].

The enzymatic activity in multilayers increased linearly with the number of layers up to 10-15 protein layers, at which point the film bioactivity became saturated. This saturation was probably due to substrate diffusion limitations into the film, that is, accessibility to the protein requires a substrate transport through the multilayer [95]. For the antigen-antibody reaction in immunoglobulin (IgG)/PSS multilayers, the activity increased up to five immunoglobu-lin layers. Compactness or openness of protein multilayers may be regulated. Thus, glucose oxidase, myoglobin, and albumin multilayer films were compact, but immunoglobu-lin/PSS multilayers had an open structure with areas as large as 100-nm in diameter unfilled in the upper layers of the film [87].

Drying of polyion films exerts unclear influences on the structure. We do not need drying for the assembly process; samples were dried for control of the assembly. From the other hand, the importance of film drying (as a separate process) is still not fully understood. The assembly with a regular film, drying at every other adsorption step, gave polar multilayers with nonlinear optical properties [99]. A similarly prepared Photosynthetic Reaction Center/PDDA multilayer demonstrated second harmonic light generation [89]. Regular immunoglobulin/PSS assembly with the film drying at every adsorption cycle was possible at a pH close to the IgG isoelectric point, which again indicates an importance of hydrophobic interactions [87].

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