Research into the interaction and electron exchange between redox enzymes and an electrode interface is important for two reasons: first, it is helpful to understand the intrinsic redox properties of proteins (1,2); and second, direct electron transfer between enzymes and electrodes is the key to the development of mediatorless, also called third-generation, enzyme biosensors. With a mediatorless biosensor, no cosubstrate is required in the recycling of the enzyme back to its active form. This concept of the mediatorless enzyme biosensor is most applicable to the oxidoreductase enzymes where, as shown in Scheme 1 for glucose oxidase, the enzyme oxidizes glucose and is reduced in the process. The recycling of the enzyme back to its active oxidized form is achieved using either oxygen in nature or another mediating species such as ferrocene (as used in many glucose meters). The ability of the enzyme

From: Methods in Molecular Biology, vol. 300: Protein Nanotechnology, Protocols, instrumentation, and Applications Edited by: T. Vo-Dinh © Humana Press inc., Totowa, NJ


GODFadh2 + Gluconolactone


Scheme 1. Reaction mechanism for glucose oxidase oxidizing glucose to gluconolactone.

to be oxidized and reduced directly at the electrode would obviate this second reaction, which has implications for reliably sensing in vivo and in other environments where cosubstrate concentration may vary.

The redox centers of most redox-active biological molecules are imbedded deep within the glycoprotein (1). For example, in the case of glucose oxidase, the closest approach between the exterior of the protein and the redox-active center flavin adenine dinucleotide (FAD) is 13 A (3). As a consequence, electrons cannot be efficiently transferred between the enzyme and the electrode and hence, mediators, or redox relays, are required. There are some exceptions, such as the peroxidase enzymes, laccase and "blue" copper protein, and azurin, in which the redox centers are located close to the surface of the protein and, hence, can be interrogated electrochemically. There have been a variety of approaches to improving the communication between the electrode and the enzyme that usually involve modifying either the electrode or the protein to allow a more intimate association between the protein and the electrode.

Pyrolytic graphite edge-plane electrodes (4) and self-assembled monolayer (SAM)-modified gold electrodes (5) have most frequently been used to achieve efficient communication to proteins. The edge planes of pyrolytic graphite contain many organic functionalities such as alcohols, phenolics, carboxylic acids, and other carbonyls. These edge planes allow rapid electron transfer while often maintaining the proteins in their functional state. Pyrolytic graphite, however, provides macroscopic surfaces on which many enzyme molecules will bind. Thus, although the communication between enzymes and the electrode is often effective, there is no control over probing individual enzymes or building up bioelectronic systems on the molecular level. However, this may be possible using carbon nanotubes (CNTs) as electrodes with which to communicate with enzymes.

Was this article helpful?

0 0
Waste Management And Control

Waste Management And Control

Get All The Support And Guidance You Need To Be A Success At Understanding Waste Management. This Book Is One Of The Most Valuable Resources In The World When It Comes To The Truth about Environment, Waste and Landfills.

Get My Free Ebook

Post a comment