Enzyme monolayer

Patterning of enzyme monolayers on a solid surface was carried out by photoactivation of immobilized monolayer of caged -biotin derivatives in selected areas. Specific oriented binding of enzyme-avidin conjugates could be readily made to the photoactivated zones [42]. Oriented immobilization of G-protein-coupled receptors on a solid surface was also made possible on a biotinylated surface by first immobilizing streptavidin, followed by the immobilization of biotinylated G-protein-coupled receptor [43]. 

Already immobilized enzyme monolayers can be inactivated with iodoa-cetate, and active monolayers can be attached de novo on top of the inactivated layers. The use of these procedures allows the construction of a large variety of structures whose spatial order can be investigated as detailed in the next section. 

The simplest situation is that represented in Figure 5.30a, where the concentration profiles of P and Q are linear in between the electrode surface and the eleventh enzyme monolayer, which is the only active layer in the film. At the plateau (see Section 6.5.3),... 

Still with an enzyme monolayer, the synthesis and current responses of a system that involves simultaneous attachment of the cosubstrate to the electrode coating are then described. The next step consists in constructing a multilayered coating constituted by successive layers of enzyme built thanks to antigen-antibody interactions. Sensing the diffusion of the cosubstrate through the film thus constructed provides evidence for spatial order and an estimate of the distances between layers. 

A supramolecular complexation between avidin and biotin-labeled enzyme results in the formation of enzyme multilayers composed of avidin and enzyme monolayers, in which each monolayer is connected through avidin-biotin complexation with each other. The thickness of the multilayers can be precisely controlled by regulating the deposition number (the thickness of each avidin plus enzyme double layer is approximately 10 nm). The enzyme multilayers are useful in preparing high... 

Parts a and b of Figure 4 illustrate that the voltammetric plateau current depends on the number of enzyme monolayers. 

The photoisomerizable enzyme monolayer electrode also revealed photoswitchable bioelectrocatalytic activity (Figure 7.10). In the presence of ferrocene carboxylic acid (5) as a diffusional electron transfer mediator, the nitrospiropyran-tethered GOx (4a) revealed a high bioelectrocatalytic activity, reflected by a high electrocatalytic anodic current. The protonated nitromerocyanine-GOx (4b) exhibited a two-fold lower activity, as reflected by the decreased bioelectrocatalytic current. By the reversible photoisomerization of the enzyme electrode between the 4a- and 4b-states, the current responses are cycled between high and low values (Figure 7.10, inset). 

Figure 3-31. Cyclic voltammograms corresponding to the photoswitchable bioelectrocatalyzed oxidation of glucose, 50 mM, in the presence of ferrocene carboxylic acid, (21), 5x 0 M, as diffusional electron mediator (a) and (c) In the presence of the SP-GOx monolayer electrode generated by the irradiation of the electrode A, > 475 run. (b) and (d) In the presence of the MRlT-GOx monolayer electrode generated by the illumination of the electrode with filtered light 320 nm < A < 380 nm. Inset cychc photoswitchable ON and OFF amperometric responses of the functionalized enzyme monolayer upon the light-induced isomerization of the interface between the SP GOx and MRI I GOx, respectively. Reproduced with permission from ref. 88. Copyright 1997 American Chemical Society.

The bioelectrocatalyzed oxidation of glucose in this system originates from the primary oxidation of the ferrocene carboxylic acid, (21), to the respective ferrocenylium cation that mediates the oxidation of the enzyme s redox center and its activation towards the oxidation of glucose. Photoisomerization of the enzyme monolayer to the MRH-GO state switched-off the bioelectrocatalytic functions of the protein monolayer, and only the electrical response of the diffusional electron mediator was observed, Fig. 3-31, curves (b) and (d). By the cyclic photoisomerization of the enzyme-monolayer electrode between the SP-GOx and MRlT-GOx states, the reversible photoswitching of the enzyme activity between ON and OFF states was demonstrated, Fig. 3-31 (inset). 

Patolsky, K, Zayats, M., Katz, E., and Willner, I. (1999) Precipitation of an insoluble product on enzyme monolayer electrodes for biosensor applications Characterization by faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance analyses. Anal. Chem. 71, 3171-3180... 

ELECTRONICALLY TRANSDUCED PHOTOCHEMICAL SWITCHING OF ENZYME MONOLAYERS... 

Electronically Transduced Photochemical Switching of Enzyme Monolayers 227... 

Tatsuma T, Okawa Y, Watanabe T. Enzyme monolayer- and bilayer-modifxed tin oxide electrodes for the determination of hydrogen peroxide and glucose. Anal Chem 1989 61 2352-2355. 

A number of biomolecules have been physically immobilised on conducting polymers [66,112, 116-119]. This is the simplest method of enzyme immobilisation. Since the binding forces involved are hydrogen bonds, van der Waals forces, etc., porous conducting polymer surfaces are most commonly used. The pre-adsorption of an enzyme monolayer prior to the electrodeposition of the polymer, [120] and two-step enzyme adsorption on the bare electrode surface and then on PPy film [121] have also been investigated. 

The interference of water absorption is eliminated when the physicochemical and dynamic approaches are used for measuring the spectra, since a water response to a perturbation differs from that of a protein (vide supra). As an alternative, the spectrum can be rendered practically free from background absorption with a polarization modulation technique. To illustrate. Fig. 7.54 shows the spectrum from PM-IRRAS of an acetylcholinesterase (AChE) (enzyme) monolayer at the air-water interface at different surface pressures [838]. The spectra were... 

Antigen-Antibody Construction of an Immobilized Enzyme Monolayer and Kinetic Analysis of the Enzymatic Catalytic Reaction... 

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