Flavins are divided into flavin adenine dinucleotide FAD , and flavin mononucleotide FMN , and they are also two-electron, two-proton redox centers Walsh As for quinones, their redox potentials are pH-dependent. In this case, the intermediate radicals are not accessible, and they are considered hydride carriers in biological systems. The difference between them is the presence of an additional phosphate on the ribose ring of the adenosine; however, their redox potentials are the same Bartlett There are different heme types, which vary according to the substitution around the porphyrin ring; consequently, the redox potential of the iron center is affected, causing the redox potentials of these groups to vary.
For iron-sulfur clusters, iron atoms are bonded to sulfur atoms on cysteine residues of the associated protein and inorganic sulfur atoms, and these function as multielectron redox centers that can pick up or release one electron at a time Beinert et al. An enzyme reaction at an electrode surface can proceed in two ways. The first approach is mediated electron transfer Figure 1 a , which is based on the utilization of redox mediators and, in this case, the enzyme catalyzes the oxidation or reduction of the mediator Cardosi and Turner , Bartlett et al.
In this type of system, the catalytic process involves the enzymatic transformation of the analyte and the mediator. In the second, in contrast, direct mediatorless electron transfer occurs Figure 1 b Tarasevich In this case, the electron is directly transferred from the active center of the enzyme to the electrode surface, which provides important information about the thermodynamics and kinetics of the biological redox process.
Because many proteins have their redox sites buried deeply in their structure, the redox center is isolated from the environment; thus, DET with bulk electrodes is hindered. In this case, the electrical communication between the enzyme and the electrode surface can be established by using charge-carriers, so-called ET mediators.
These agents are artificial electron acceptor or donor molecules able to shuttle electrons from the redox center of the enzyme to the electrode and vice versa Katz et al. An ideal redox mediator should provide a rapid reaction with the enzyme, exhibit reversible electrochemistry large rate constant for the interfacial ET at the electrode surface , be stable in the oxidized and reduced forms under the working conditions, have a low overpotential for regeneration, and do not participate in side reactions during ET.
Furthermore, the redox potential of the mediator should be more positive for oxidative biocatalysis and more negative for reductive biocatalysis, compared to the redox potential of the enzyme active site Chaubey and Malhotra For the selection of a suitable mediator, some factors must be considered, such as the redox potential, stability, and solubility under the working conditions, and the properties of the enzyme and the mediator Kavanagh and Leech Several designs of mediated bioelectrochemical systems have been developed.
These systems can involve from soluble enzymes with diffusional electron mediators to sophisticated architectures with multistep mediated ET. The simplest system configuration involves the enzyme and mediator in solution, i. In this case, the mediator reacts with the enzyme in the bulk solution and diffuses to the electrode, where it is regenerated Kavanagh and Leech This kind of system is useful for studying the enzyme-mediator interactions, for example, the influence of structure mediators in the redox reaction with the enzyme Forrow et al.
Other systems employ heterogeneous electron mediation Patolsky et al. Alternatively, both enzyme and mediator can be incorporated into the electrode surface Reuillard et al. These systems produce high currents because the enzyme and mediator are present in high concentration at the electrode surface. An alternative approach to the mediated system is the use of soluble enzymes functionalized with electron mediators.
For example, GOx has been covalently modified with ferrocene, osmium, and ruthenium complexes by the formation of bonds with lysine or histidine residues Katz et al. The utilization of mediated ET system must be carefully considered. Although it facilitates the electrical connection between enzymes and electrodes, providing biofuel cells with large currents, power outputs, and small voltage losses, resulting in sensitive biosensors, the use of mediators can limit the application of bioelectrochemical devices.
This is because many redox mediators are toxic, which precludes the implantation of these devices in vivo Falk et al. Moreover, the miniaturization of devices with mediated ET processes is quite complicated because the compartmentalization of the device using membranes is necessary. Nowadays, many researchers are interested in achieving DET between an electrode and the active center of an enzyme, and this is very important for the development of next-generation enzyme biosensors and biofuel cells Willner In addition, non-mediated bioelectrochemistry at solid electrodes has been developed as a potentially powerful method for mechanistic studies of redox proteins Frew and Hill DET has been observed in redox proteins where the redox center is close to the surface of the protein, such as cytochrome c Eddowes and Hill and ferredoxin Armstrong et al.
However, for proteins such as GOx where the prosthetic group, FAD, is deeply embedded within a protective protein shell, it is difficult to observe this type of charge transfer. An immobilized enzyme capable of DET will allow the electrochemical measurement of the enzyme substrate without the addition of any mediator to analyze the ET process Zhao et al.
In addition, it has been suggested that DET may proceed most easily to or from electrode surfaces when the environment is similar to the native environment of the redox protein Zhao et al. Thus, obtaining DET between enzymes and electrode surfaces is important, once this process could be applied to the study of enzyme-catalyzed reactions in biological systems and in the investigation of the mechanisms of redox reactions at enzymes molecules Cai and Chen They showed the reversible electrochemistry, using cyclic voltammetry CV , of cytochrome -c on bipyridyl-modified gold and tin-doped indium oxide electrodes.
In this case, it was shown that laccase-modified Berezin et al. Many redox proteins have demonstrated efficient DET reactions; however, these proteins have no intrinsic catalytic activity but act as ET components in biochemical pathways, e. On the other hand, efficient DET reactions with electrodes have been reported for few redox enzymes, e.
In principle, two experimental approaches could establish if DET occurs between the enzyme and the electrode surface: indirect evidence based on the catalytic response current in the presence of the substrate and direct evidence from the independent electrochemical activity of the redox cofactor in the absence of the substrate.
Many enzymes with known DET properties contain a metallocenter at the active site, e.
However, there are some enzymes with DET properties that contain only an organic cofactor, such as flavin Figure 2 b Wilson and Turner For DET occurrence in the redox proteins, there are some prerequisites. According to Marcus theory Marcus , the DET rate between two redox sites will depend on three factors: the reorganization energy, which is divided into the inner and outer contributions, where the first is related to the energy necessary to modify bond distances and the second is related to the energy necessary to reorganize the solvent; the potential difference between the redox centers; and the distance between the redox sites Carter et al.
Thus, ET between large redox proteins and the electrode surface is usually slow and sometimes difficult to achieve Heller and Degani because the redox center is deeply embedded in the protein structure.
As mentioned above, in many cases, a direct enzymatic electrochemical reaction is difficult because of factors such as the way in which the enzyme is adsorbed on the electrode surface, which could result in the denaturation and loss of electrochemical activity and bioactivity. Moreover, the large size of the enzyme results in the inaccessibility of the redox center, making it difficult to obtain DET Cai and Chen As cited above, electron tunneling from the enzymatic redox center to the electrode surface and vice versa can be described by the Marcus-Hush-Chidsey formalism.
Subsequently, Chidsey showed the dependence of the ET rates on distance at the electrode, and the dependence of the ET with the temperature at the metal-electrolyte interface Chidsey Applying the Marcus-Hush-Chidsey model to bioelectrochemistry, it is possible to conclude that the ET depends upon the enzyme structure, the position of the redox center inside the protein structure, the enzyme orientation, and the ET distance, which varies exponentially Cooney et al. Equation 3 describes a non-adiabatic ET, which occurs for the most protein processes Luz et al. Here, h is the Planck constant, k B is the Boltzmann constant, and T is the temperature.
Here, k max is the asymptotic value of the rate constant at high overpotential, which is given by Equation 5. Thus, one possibility to improve the DET between the electrode surface and the enzyme is to shorten the distance between the active center and the electrode by modifying the electrode surface or the protein structure, as will be described in Section Protein-electrode interfaces.
Snider, Sarah M. This system is composed by a conventional electrochemical cell connected in a mass spectrometer and the bioelectrochemical cell had an FCF with immobilized ADH as working electrode, the electrical contact was provided by a gold ring and wire, and the working electrode is supported over a polytetrafluoroethylene PTFE membrane that allows only the passage of gaseous and volatile compounds. This acid cleaves the glycosidic bonds, without changing the protein core, maintaining the enzyme activity Edge et al. Some studies point out the lack of antibacterial effects of the GO 9 , while on the contrary, other studies affirm that the GO materials destroy cellular integrity Using examples from real biofilm research to illustrate the techniques used for electrochemically active biofilms, this book is of most use to researchers and educators studying microbial fuel cell and bioelectrochemical systems. Energy Environ.
Another approach that has been utilized is the incorporation of nanoparticles to the electrodes Zhang et al. This approach is promising because nanoparticles have high specific surface areas and excellent biocompatibility and conductivity. For example, gold nanoparticles can adsorb redox enzymes without loss of the enzyme activity Hayat , and the nanoparticles act as conduction centers, facilitating the transfer of electrons. Zhao et al.
This could be attributed to the oxygen-containing groups Musameh et al. Thus, both mediated and direct ET have advantages and disadvantages, and it is necessary to analyze the goal of the study and its applications to choose the most suitable method. Some parameters that govern ET can be modulated, such as the distance between the redox center of the protein and the electrode, the Fermi level of the electrode, and the protein orientation on the surface.
Thus, the electrode interface plays a key role with regard to ET between the enzyme and the electrode surface. Thereby, to achieve charge transfer and bioelectrocatalysis of some enzymes, it is necessary to modify the electrode or protein structure. Nanoparticles, carbon nanotubes, graphene, among others have been used to modify electrode surfaces successfully. Regarding the modification in enzyme structure, deglycosylation and oligomerization procedures have been employed to improve the ET and bioelectrocatalysis.
For instance, nanoparticles Katz and Willner , Xiao et al. Concerning nanoparticles, gold nanoparticles AuNPs can be functionalized with organic molecules containing thiol groups Xiao et al. Therefore, the presence of AuNPs decreases the effective ET distance between the redox center of Cyt- c and the electrode surface. It is important to note that the scheme is merely illustrative and does not use a realistic length scale.
Carbon nanotubes are interesting materials for bioelectrodes because of their attractive properties, such as their inherently high surface area, tubular structure, and electrocatalytic properties Guiseppi-Elie et al. In addition, carbon nanotubes approximate a redox active center, as for cytochrome -c Davis et al. Another attractive property of carbon nanotubes is the possibility of aligning CNT assemblies on the electrode surface, wherein the length and the density of the assemblies can be controlled Gooding et al.
Graphene is another carbon nanomaterial that has been applied to enzymatic ET studies, and it has extraordinary electron transport properties Li et al. Graphene can promote ET in a matrix and facilitate the DET process between the redox center of protein and the electrode Shan et al. Other strategies to improve the ET have been used, such as the modification of surface with polymers, the functionalization of the electrode surface, the use of redox mediators, and the use of mesoporous materials and composites. Conducting and redox polymers have been applied to bioelectrodes since the s Degani and Heller , Foulds and Lowe , , Pandey to improve the communication between the enzyme and electrode, thus resulting in fast ET.
Redox mediators are used to promote effective ET because some oxidoreductases enzymes are not able to transfer electrons by themselves Moehlenbrock and Minteer Furthermore, many low-molecular-weight redox active compounds have been used Chaubey and Malhotra , such as methylene blue McCord and Fridovich , toluidine blue O Boguslavsky et al. Some mesoporous materials have also been used to study ET and develop biosensors Dai et al.
Composites have been studied as a platform to enhance the performance of bioelectrodes, particularly nanocomposites have been utilized to improve the amperometric response of bio-relevant molecules such as dopamine, hydrogen peroxide, or NADH Le Goff et al. The modification and functionalization of electrodes can improve the ET because these strategies result in the excellent adsorption of the enzyme on the electrode surface. In this context, the modification and functionalization of flexible carbon fibers FCF has been studied to prepare an optimal electrode for the investigation of bioelectrochemical processes Martins et al.
This modification provided a bioelectrode with unprecedented electrocatalytic performance. Reprinted from de Souza et al. ADH was immobilized on the pristine FCF and on the FCF functionalized with quinone-like groups, and the performances of the electrodes were compared using electrochemical measurements in the presence of several ethanol concentrations, as shown in Figure 5. All measurements were carried out in N 2 -saturated, 0.
For the cyclic voltammograms, the scan rate was 50 mVs -1 , and, for chronoamperometry, the applied potential was 0. Reprinted from Pereira et al.
As shown, after the modification of the FCF surface, there is an improvement in the bioelectrocatalysis, and the current densities increase around 10 times. This indicates that the modification of electrode surfaces could enhance the communication between the electrode and the enzyme.
Besides the modification of the electrode surface, another possibility to improve the charge transfer is the modification of the protein structure. Mano and coauthors Courjean et al.
Deglycosylation corresponds to the cleavage of the glycans of the protein structure without changing the protein core Courjean et al. The glycans are bonded to the protein backbone by glycosidic and amidic bonds, which could be cleaved by enzymatic or chemical routes. Thus, after this procedure, the enzyme could be utilized as the biocatalysts in solid electrodes with the active center closer to the surface, resulting in an increased faradaic current and improving the performance of the bioanodes in biofuel cells.
Enzymatic procedures Courjean et al. Enzymatic routes allow for mild conditions, but they are specific for certain glycans. On the other hand, chemical methods Patel et al. Moreover, they can remove all the glycans under appropriate reaction conditions. In this case, the reactants cleave the glycosidic bonds involving mainly neutral sugars.
Trifluoromethanesulfonic acid TFMS is a strong acid that has been utilized for the chemical deglycosylation of glycoproteins. This acid cleaves the glycosidic bonds, without changing the protein core, maintaining the enzyme activity Edge et al. For example, for the deglycosylated horseradish peroxidase HRP , a recombinant enzyme, it was showed a much higher rate of heterogeneous DET than for native one. In addition, the percentage of adsorbed enzyme molecules oriented for DET was increased compared to the wild-type HRP.
The glycosylation could be considered as the reason for the absence of any electrochemical response of laccase from Coriolopsis fulvocinerea under anaerobic conditions; that is, it increases the distance of the electron tunneling between the laccase and conducting carbon Shleev et al.