Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors
Introduction
Modern analytical chemistry turns to the step when principally new fundamental approaches and smart materials are developed in order to solve regular analytical problems. One of the intriguing phenomena known for the last few decades is bioelectrocatalysis with the particular case of direct electron transfer (DET) between the electrode material and redox active biomolecules. The very first reports on DET with a redox active protein were published already in 1977 when Eddows and Hill [1] and Yeh and Kuwana [2] independently showed that cytochrome con gold and tin doped indium oxide electrodes, respectively, showed virtually reversible electrochemistry as revealed by cyclic voltammetry. Cytochrome cis a small redox protein which is active in biological electron transfer (ET) chains but has no enzyme properties. These first publications were soon followed in 1979 by reports from Russian groups giving evidence that DET was possible also for larger redox proteins with enzymatic activity (oxidoreductases or ‘redox enzymes’). They showed that, in the presence of the enzyme substrate (molecular oxygen), laccase modified carbon [3] and (hydrogen peroxide) peroxidase modified carbon electrodes [4] revealed DET. These findings occurred some 10 years after the first papers on enzyme based amperometric biosensors were published [5]. The electronic coupling between redox enzymes and electrodes for the construction of analytical detection devices (enzyme electrodes or biosensors) has, however, in most cases, not been based on DET but rather on the electroactivity of the enzyme substrate or product (first generation biosensors) or through the use of redox mediators (second generation biosensors) most typically illustrated by numerous biosensors based on glucose oxidase [6], [7], [8]. The drawbacks with first generation biosensors, such as too high an applied potential, put the focus on the use of mediators, small redox active molecules that could diffuse in and react with the active site of the enzyme and diffuse out and react with the electrode surface, thus shuttling the electrons between the enzyme and the electrode [7], [8], [9], [10]. The use of mediators made it possible to decrease the applied potential for biosensors based on glucose oxidase [11] and many other hydrogen peroxide producing oxidases decreasing the influence from bias signals caused by electrochemically easily oxidisable interfering compounds present in real samples. However, the use of mediators also opened up the possibility for the use of other groups of redox enzymes, e.g., various dehydrogenases, peroxidases and even whole cells or organelles [12], [13], [14], [15], [16], [17]. Further progress in the development of second generation biosensors was achieved with the use of flexible polymers onto which mediating functionalities were covalently bound [7], [18]. However, the redox mediators used in conjunction with redox enzymes are in no way selective but rather general redox catalysts facilitating not only the ET between electrode and enzyme but also various interfering reactions. Commercial biosensors are based on either first or second generation biosensors and are exemplified with, e.g., Yellow Springs and MediSense glucose electrodes, respectively.
For biosensors based on DET, third generation biosensors[19], the absence of mediators is the main advantage, providing them with superior selectivity, both because they should operate in a potential window closer to the redox potential of the enzyme itself, and therefore, less prone to interfering reactions [8] and also because of the lack of yet another reagent in the reaction sequence. Another attractive feature of the systems based on DET is the possibility of modulating the desired properties of an analytical device using protein modification with genetic or chemical engineering techniques on one hand [20], [21], [22], and novel interfacial technologies on the other hand [23], [24].
In this context, chemical conversion of a single-centre redox enzyme into a two-centre one by introducing a mediator molecule (pyrrolo quinoline quinone (PQQ)) located half way through a synthetic ET pathway is also of particular practical importance [25], [26]. The procedure was demonstrated to result in interference free electrodes probably due to the fact that an artificially introduced redox centre is shielded by the protein as well as due to the rapid electron exchange with the electrode. The approach requires, however, a high skilled manipulation for unfolding–modifying–refolding of the enzyme.
Efficient DET reaction has been reported for a restricted number of redox enzymes [19], [27], [28] (see Table 1). The vast majority of these redox enzymes contain metallocentres and heme in particular. In most of these cases, DET was proven in the presence of the enzyme substrate as a catalytic current; in still fewer cases, independent electrochemical proofs of DET have been shown in the absence of substrate.
Research on DET based reactions in our laboratory has been going on since our first report on DET between graphite electrodes modified with adsorbed horseradish peroxidase (HRP) [33] and it is now a central theme from both fundamental and applied aspects [8], [32], [71]. Below follows a compilation of the current on-going research in this direction focused on heme containing proteins and enzymes.
Section snippets
Heme
Heme is a molecule which forms a number of reduced and oxidised states. Moreover, its electrochemical characteristics, e.g., formal potential (E0′) for its redox conversion between Fe2+ and Fe3+, can be varied over a wide range of potentials by the protein environment, e.g., from −0.27 V versus SHE (for HRP [72]) to 0.26 V (for cytochrome c[73], [74]). By itself, heme exhibits various catalytic properties, which drastically change when incorporated into a proteinaceous environment. This creates
Cytochrome c
Cytochrome c is a heme-containing redox protein active in ET pathways, e.g., in the respiratory chain in the mitochondria [79]. Under physiological conditions, it does not react with molecular oxygen but is readily oxidised by its natural redox partners cytochrome oxidase and cytochrome c peroxidase. As mentioned above, cytochrome c was the very first redox protein to be studied electrochemically and it has remained one of the most popular heme-proteins in electrochemical studies and
Microperoxidase
The interfacial interactions and protein orientation are the most important parameters restricting the achievement of maximum ET rates. One of the approaches for solving the orientation problem is the maximum exposure of the catalytic site to the electrode surface, or as stated above, minimising the distance between electrode surface and the redox active site. Electrochemistry of microperoxidase could be considered as the best example of such an approach. Microperoxidase is produced via
Peroxidases
HRP is one of the most widely used enzymes for analytical purposes and biosensors in particular [32]. Mainly HRP but also other peroxidase modified electrodes have been used in a straightforward mode for the detection of hydrogen peroxide and other hydroperoxides and they have also been used in conjunction with hydrogen peroxide producing oxidases for the measurement of the concentration of the oxidase substrate [32]. Both direct and mediated ET approaches have been used for electronic coupling
Bifunctional heme-enzymes
Many native redox enzymes contain a number of prosthetic groups, which are linked by ET pathways. These enzymes provide a unique possibility of studying both heterogeneous and internal ET and its modulation by the protein/electrode interface. Our recent work was devoted to the electrochemistry of cellobiose dehydrogenase (CDH). CDH is an extracellular enzyme produced by a number of cellulolytic wood degrading fungi. The most well studied is the enzyme from the white rot fungus Phanerochaete
Acknowledgements
The authors thank the following organisations for financial support: The Swedish Natural Science Research Council (NFR), The Swedish Royal Academy of Science (KVA), The European Commission (BIO4-CT97-2199 and ERBIC15CT961008), and the Crafoord Foundation. The authors also thank Dr. Gunnar Henriksson, Dept. of Biochemistry, Uppsala University, for donating CDH, Dr. Motomasa Tanaka, Kyoto University, Japan for the HRP mutants, Dr. Ivan Yu. Sakharov, Santander Industrial University, Colombia, for
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