Deposition protein layers

Electrode surfaces modified with a multilayered surface architecture prepared by a layer-by-layer repeated deposition of several enzyme mono-layers show a modulated increase of surface-bound protein with a subsequent increase in output current, which is directly correlated with the number of deposited protein layers. The versatility of this approach allows alternate layers of different proteins for the manufacture of electrode surfaces, which are the basis for multianalyte sensing devices with multiple substrate specificities. The surface chemistry used for the manufacture of multilayered electrode surfaces is similar to that previously described for the preparation of affinity sensors, and is based on the stabilization of self-assembled multilayer assemblies by specific affinity interactions, electrostatic attraction, or covalent binding between adjacent monolayers. 

Dorson, W. J., Jr., Cotter, D. J., and Pizziconi, V. B. (1975). Ultrafiltration of molecules through deposited protein layers. Trans. ASAIO 21, 132. 

The other method of monolayer transfer from the air/water interface onto solid substrates is illustrated in Figure 2. This method is called the Langmuir-Schaefer technique, or horizontal lift. It was developed in 1938 by I. Langmuir and V. Schaefer for deposition of protein layers. Prepared substrate horizontally touches the monolayer, and the layer transfers itself onto the substrate surface. The method is often used for the deposition of rigid monolayers and for protein monolayers, hi both cases the apphcation of the Lang-muir-Blodgett method produces defective films. 

The Langmuir-Blodgett (LB) technique was successfully applied for the deposition of thin protein layers (Langmuir and Schaefer 1938, Tiede 1985, Lvov et al. 1991). LB organization of protein molecules in film not only preserved the structure and functionality of the molecules, but also resulted in the appearance of new, useful properties, such as enhanced thermal stability (Nicolini et al. 1993 Erokhin et al. 1995). 

The LB technique was chosen for covering the spheres because it was shown to provide enhanced thermal stability of many types of proteins in deposited layers (Nicolini et al. 1993, Erokhin et al. 1995, Antolini et al. 1995), which no other technique is able to achieve. Since only the upper protein layer is involved in the catalytic activity, no special attention was paid to check whether the deposited layer is a monolayer or multilayer. However, the samples were thoroughly washed to remove protein molecnles not bound covalently to the sphere surface, since during the functional test these molecules could contribute to the measured apparent catalytic activity. 

The described procedure allows one to deposit protein, in particular, enzyme, LB films onto the surface of small spheres. Deposited multilayer film was washed in order to leave at the surface only a layer covalently attached to the activated surface. The enzyme... 

Troupe and his coworkers reported a two-step method to electrochemically deposit GOx enzyme on a diamond film electrode (Troupe et al. 1998). In the first step, the GOx and BSA were dissolved in a buffer with pH = 7, greater than their isoelectric points. The BDD electrode was immersed into the solution and biased at 3.5 V for 2 h. Therefore, the negatively charged proteins (GOx and BSA) were drawn to the positively charged electrode. In the second step, the glutaraldehyde (GA) cross-links the protein layer forming a water insoluble barrier. 

Note A , area of unblocked membrane (m ) Aq, initial area of unblocked membrane (m ) Cb, bulk concentration (g-L ) /, fractional amount of total foulant contributing to deposit growth 7b> filtrate flux within the blocked area (m s ) Q, volumetric flow rate (m s ) Tp, radius of membrane pore (m) Rm, resistance of the clean membrane (m ) Rp, resistance of the deposit (m ) R, specific protein layer resistance (mkg ) t, filtration time (s). Greek letters a, pore blockage parameter (m kg ) J3, pore contriction parameter (kg) 5, membrane thickness (m). 

A method first described around 1980 has remained popular - drying a slurry of support material (commonly celite) in an enzyme solution. The resulting powder necessarily contains both solid support and enzyme. However, the link between them is usually very weak, and the method is now usually described as co-drying or deposition rather than immobilization. At least some of the enzyme is probably present as large aggregates, not close to the support. My impression is that catalytic activity is usually poorer than with enzymes immobilized by methods more likely to produce an even protein layer. 

The first event that generally occurs after blood contacts a polymer surface is the formation of a protein layer at the blood-polymer interface (1). The formation of this protein layer is followed by the adherence of platelets, fibrin, and possibly leukocytes (2). Further deposition with entrapment of erythrocytes and other formed elements in a fibrin network constitutes thrombus formation. The growth of the thrombus eventually results in partial or total blockage of the lumen unless the thrombus is sheared off or otherwise released from the surface as an embolus (3). Emboli can travel downstream, lodge in vital organs, and cause infarction of tissues. The degree to which the polymer surface promotes thrombus formation and embolization, hemolysis, and protein denaturation determines its usefulness as a biomaterial (4). 

The bioadhesion mechanism is not well defined. The Hterature indicates that bioadhesion takes place in three steps the deposition and attachment of a protein layer, followed by cell deposition. The important parameter corresponds to the formation of a water monolayer on the substrate (Fig. 12.1). 

BiocompatibiHty is shown to be correlated to the surface properties and to the possibility of deposition and attachment of a protein layer, i.e., a biofilm. The... 

Blood responses. Blood is the fluid which transports body nutrients and waste products to and from the extravscular tissue and organs, and as such is a vital and special body tissue. The major response of blood to any foreign surface (which includes most extravascular surfaces of the body s own tissues) is first to deposit a layer of proteins and then, within seconds to minutes, a thrombus composed of blood cells and fibrin (a fibrous protein). The character of the thrombus will depend on the rate and pattern of blood flow in the vicinity. Thus, the design of the biomaterial system is particularly important for cardiovascular implants and devices. The thrombus may break off and flow downstream as an embolus and this can be a very dangerous event. In some cases the biomaterial interface may eventually "heal" and become covered with a "passive" layer of protein and/or cells. Growth of a continuous monolayer of endothelial cells onto this interface is the one most desirable end-point for a biomaterial in contact with blood. Figure 10 summarizes possible blood responses to polymeric biomaterials. 

Studies of typical nanomaterials (soil mineral components, adsorbents, silica gels with deposited proteins, so called smart surfaces, latexes, synthetic zeolites modified by ions, MCM-41 molecular sieves) were made earlier by the author of this chapter [11-17]. At present our research focuses on studies of surface properties (e.g. adsorption capacity), total heterogeneity (energetic and geometrical) of surface layers, as well as structures and phase transformations of... 

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