Enzymes, molecular interfacing

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. 

Molecular Interface for Electron Transfer of Electrode-bound Redox Enzymes... 

These progresses in electron transfer of enzymes have led us to conclude that a molecular level assembly should be designed to facilitate electron transfer at the interface between an enzyme molecule and an electrode. Such a molecular level of assembly at the interface may be termed "molecular interfaces" [8-10]. 

There are several molecular interfaces for redox enzymes to promote electron transfer at the electrode surface (Fig.6). 

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. 

Fig. 10 Schematic illustration of the molecularly interfaced enzyme on the electrode surface...

Electrochemical communication between electrode-bound enzyme and an electrode was confirmed by such electrochemical characterizations as differential pulse voltammetxy. As shown in Fig. 11, reversible electron transfer of molecularly interfaced FDH was confirmed by differential pulse voltammetry. The electrochemical characteristics of the polypyrrole interfaced FDH electrode were compared with those of the FDH electrode. The important difference between the electrochemical activities of these two electrodes is as follows by the employment of a conductive PP interface, the redox potential of FDH shifted slightly as compared to the redox potential of PQQ, which prosthetic group of FDH and the electrode shuttling between the prosthetic group of FDH and the electrode through the PP interface. In addition, the anodic and cathodic peak shapes and peak currents of PP/FDH/Pt electrode were identical, which suggests reversibility of the electron transport process. 

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]. 

MOLECULAR SENSING DEVICES WITH MOLECULAR INTERFACES ENZYMES... 

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. 

Investigation on the molecular interfacing of redox enzymes yields the following important findings. The molecular-interfaced redox enzymes showed the potential dependency of enzyme activity. The enzyme was inactive when the electrode potential was set below a certain threshold. In contrast, the enzyme activity increased with an increase in the electrode potential above the threshold. The activity of the molecular-interfaced enzyme is reversibly modulated by changing the electrode potential. 

Fig.27 Potential dependency of the enzyme activity of the molecularly interfaced glucose oxidase...

The response current extremely decreased in the potential range above 0.6V probably due to an irreversible inactivation of FDH. The possible reasons for the inactivation at higher potentials might be (1) conformational change of FDH in such a manner as the enzyme loses its prosthetic group PQQ, and (2) a drastic change in pH of the molecular interface that causes the enzyme inactivated. 

It should be emphasized that the enzyme activity of the molecular interfaced FDH was reversibly controlled by electrode potential in the potential range from 0.1 to 0.6V. It is also noted that the enzyme can be activated and inactivated at a threshold potential of 0.07V which corresponds to the redox potential of the prosthetic group PQQ. 

In Fig.26, the energy correlation is schematically presented. The potential-controlled modulation of the molecular-interfaced enzymes may be interpreted by Fig.26. The enzyme and its substrate molecule have their intrinsic redox potentials. The redox potentials of oxidases and dehydrogenases are determined by an electron transferring molecule, i.e. a cofactor such as FAD, which is located at the active site of the enzyme. Due to potential gradient, an electron can be transferred from the substrate molecule to the active site of the enzyme, if the substrate molecule is accepted by the molecular space of the enzyme active site. However, the electron transfer between the active site of the enzyme and the electrode is regulated by the electrode potential, even if the molecule wire could be completed. It should be reasonable that the enzyme activity is electrically modulated at a threshold of the redox potential of the enzyme. 

Several protein assemblies have successfully been fabricated on the solid surfaces sifter the bioinformation transduction. These include the following molecular systems molecularly interfaced redox enzymes on the electrode surfaces, calmodulin / protein hybrides, and ordered antibody array on protein A. These protein assemblies find a wider application in various fields such as biosensors, bioreactors, and intelligent materials. 

By co-immobilizing tyrosinase with a serine esterase on a gold electrode, it is possible to establish a multistep reaction pathway that allows the activity of the esterase to be determined indirectly via measurement of o-quinone reduction at the electrode. The molecular architecture of a bi-enzyme sensor interface is shown schematically in Figure 57.13. 

Aizawa has studied an enzyme (metal) electrode based on immobilized GOD, which is a redox enzyme [66]. Interfacing in this type of biosensor requires facilitation of electron transfer from the active site of the enzyme protein to the metal electrode. Various methods have been used, such as electron mediators, electron promoters, and molecular wires, to accomplish this purpose. For example, a conducting polymer, polypyrrole, formed by electropolymerization on the electrode surface, was used to form electron wires which can facilitate electron transfers to and from the metal electrode. 

Khan, G.F, E. Kobatake, H. Shinohara, Y. Ikariyama, and M. Aizawa. 1992. Molecular interface for an activity controlled enzyme electrode and its application for the determination of fiiictose. Anal... 

Aizawa, M., T. Haruyama, G.F. Khan, E. Kobatane, and Y. Ikariyama. 1994. Electronically modulated biological functions of molecular interfaced enzymes and living cells. Biosens Bioelectron 9... 

Few redox enxymes undergo reversible electron transfer on the electrode surface primarily due to steric hindrance of the redox centers pf these enzymes. Several molecular interfaces have been designed to promote electron transfer of electrode-bound redox enzymes. 

The authors have successfully interfaced fructose dehydrogenase and alcohol dehydrogenase on the electrode surface with conducting polymer of polypyrrole, which could cause these enzymes to make an electron transfer with retaining their enzymatic activity. The molecular-interfaced redox enzymes will find their application in fabricating biosensors that respond specifically to the corresponding substrates in current. 

The performance of the PP-interfaced electrode was controllable as far as the applied potential was in the range from 0.1 V to 0.6 V without any deterioration of the enzyme. Since PP is doped state in this potential range, the y-conjugated polymer works as a molecular interface which carries electron between a protein molecule and an electrode. 

Molecular-interfaced alcohol dehydrogenase with NAD and its response to ethanol. It has been difficult to incorporate dehydrogenases that are coupled with NAD(P) into amperometric enzyme sensors owing to the irreversible electrochemical reaction of NAD. We have developed a molecular-interfaced alcohol dehydrogenase on the surface of an electrode on which NAD is electrochemical 1 y regenerated within a membrane matrix. 

Electronic communication of fructose dehydrogenase (FDH) with a Pt electrode was accomplished through the conducting polymer molecular interface. Electrons were reversibly transferred between the active center of FDH and the electrode surface when the electrode potential was properly controlled. The enzyme activity of the molecular-interfaced FDH was found to be modulated in the presence of D-fructose by the electrode potential. Electronic communication of alcohol dehydrogenase (ADH) with a Pt electrode was also accomplished in the... 

AIZAWA ET AL. Molecular Interfacing of Enzymes on Electrode Surface 313... 

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