Enzyme monolayers, electronically

Redox enzymes have been assembled in a monolayer on the solid surface by a potential-assisted self-assembling method as well as a thiol-gold selfassembling method. These enzymes are electronically communicated with the solid substrate through a molecular interface of conducting polymer and a covalently bound mediator. Electron transfer type of enzyme sensors have been fabricated by the self-assembling methods. 

The polypyrrole molecular interface has been electrochemically synthesized between the self-assembled protein molecules and the electrode surface for facilitating the enzyme with electron transfer to the electrode. Figure 9 illustrates the schematic procedure of the electrochemical preparation of the polypyrrole molecular interface. The electrode-bound protein monolayer is transferred in an electrolyte solution containing pyrrole. The electrode potential is controlled at a potential with a potentiostat to initiate the oxidative polymerization of pyrrole. The electrochemical polymerization should be interrupted before the protein monolayer is fully covered by the polypyrrole layer. A postulated electron transfer through the polypyrrole molecular interface is schematically presented in Fig. 10. 

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). 

ELECTRONICALLY TRANSDUCED PHOTOCHEMICAL SWITCHING OF ENZYME MONOLAYERS... 

Electronically Transduced Photochemical Switching of Enzyme Monolayers 227... 

From the great number of oxidoreductases used to modify enzymatic BFC electrodes only a minority is capable of DET, which reduces the number of fuels and oxidants (Table 1). The substrate specificity of enzymes redners half-cell separation by e.g., membranes unnecessary. DET between enzyme and electrode also stops the need for soluble redox mediators to shuttle electrons between enzyme and electrode. This results in the possibility to design membraneless, non-compartmentalized enzymatic BFCs with a simple architecture. However, so far achieved DET currents are lower than MET currents, because usually only enzyme monolayers can be contacted. Strategies to improve the current density aim at the use of high surface area electrode materials like CNTs, AuNPs etc. or the layer-by-layer approach... 

Enzyme monolayers can be electrically contacted by using electron mediators... 

One of the key technologies required for fabricating biomolecular electronic devices concerns with molecular assembly of electronic proteins such as redox enzymes in monolayer scale on the electrode surface. Furthermore the molecularly assembled electronic proteins are required to be electronically communicated with the electrode. Individual protein molecules on the electrode surface should be electronically accessed through the electrode. 

In contrast to the molecular wire of molecular interface, electron mediators are covalently bound to a redox enzyme in such a manner as an electron tunneling pathway is formed within the enzyme molecule. Therefore, enzyme-bound mediators work as molecular interface between an enzyme and an electrode. Degani et al. proposed the intramolecular electron pathway of ferrocene molecules which were covalently bound to glucose oxidase [ 4 ]. However, few fabrication methods have been developed to form a monolayer of mediator-modified enzymes on the electrode surface. We have succeeded in development of a novel preparation of the electron transfer system of mediator-modified enzyme by self-assembly in a porous gold-black electrode as schematically shown in Fig.12 [14]. 

Electron Transfer Type of Dehydrogenase Sensors To fabricate an enzyme sensor for fructose, we found that a molecular interface of polypyrrole was not sufficient to realize high sensitivity and stability. We thus incorporated mediators (ferricyanide and ferrocene) in the enzyme-interface for the effective and the most sensitive detection of fructose in two different ways (l) two step method first, a monolayer FDH was electrochemically adsorbed on the electrode surface by electrostatic interaction, then entrapment of mediator and electro-polymerization of pyrrole in thin membrane was simultaneously performed in a separate solution containing mediator and pyrrole, (2) one-step method co-immobilization of mediator and enzyme and polymerization of pyrrole was simultaneously done in a solution containing enzyme enzyme, mediator and pyrrole as illustrated in Fig.22. 

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