3.3.1. Electrode Functionalization
Several methods have been developed to add surface chemical functions to planar electrodes [
78] (
Figure 4). Concerning gold surfaces, the most widely used functionalization is achieved through self-assembled monolayers (SAMs). SAMs are usually formed by spontaneous adsorption of sulfur. Alkanethiols, or dialkyl disulfides or sulfides possess high affinity for the surface of gold (but also for platinum or silver), with a bond energy of RS-Au of ∼40 kcal·mol
−1 [
79]. This adsorption results in well-defined organic surfaces with desirable and alterable chemical functionalities, controlled simply by changing the terminal group of the thiol chain. The modification of a gold electrode with SAMs provides a choice of positive/negative/hydrophobic terminal functional groups such as –COOH, –NH
2, –SO
3H, –CH
3, and –OH. In addition, the knowledge of the pKa of the terminal moieties allows chemical control on the electrode surface. As examples, pKa values of 6.0 [
80] and 6.9 [
81] for SAMs of 11-mercaptoundecanoic acid (MUA) and 4-aminothiophenol (4ATP) groups on gold were determined, respectively. Dependence of the pKa of –NH
2-based SAMs on thiol chain length was also reported [
82].
Their ease of preparation from millimolar thiol solutions makes SAMs attractive candidates for surface tailoring. A surface coverage of the order of pmol·cm
−2 is generally achieved and adsorption stops at the level of monolayer coverage [
84]. This surface coverage, as well as compactness and organization of the layer, shows, however, dependence on the type of thiol molecules, nature of the metal surface, immobilization time, and thiol concentration [
85]. Longer adsorption time and longer thiol chains are expected to provide a more organized SAM layer by decreasing pinhole defects or conformational defects in the alkane chains [
86,
87]. Long-chain thiols are expected to form well-organized assemblies due to stabilizing van der Waals interaction along the adsorbed chain. On the contrary, presence of long chains may screen their mobility in the solution and, hence, their accessibility to the metal surface. It was recently proposed that fast and repeated changes on applied potential pulses relative to potential of zero charge cause an ion stirring effect [
88] which has an influence on the SAM formation kinetics. Immobilization of biomolecules on SAMs has been critically reviewed [
89]. The possibility to prepare mixed SAMs is an added advantage, where homogeneous or separated phases of different thiols may allow the site-specific binding of enzyme to the surface [
90].
Besides this, silane-based SAMs on ITO electrodes have been used owing to their simple preparation, good reproducibility, and high stability [
91]. ITO, being low cost, highly stable, and transparent, is a very useful material as an electrode due to its electrical and optical properties [
92] and plays an important role especially for biosensor technology [
93]. The silane-based chemicals act as cross-linkers because they contain Si–O bonds which react with the surface hydroxyl groups of ITO where the end groups of silane act as an immobilization matrix for biomolecules.
However, the use of SAMs for redox enzyme immobilization also raises critical issues. The first is linked to the ET process through the SAM itself, which can become a limiting step and control the whole ET process. As developed above, the efficiency of ET, given by the ET rate constant k
ET, is found to be a function of the distance d between the electrode surface and the redox species. A study related to the kinetics of ET between a gold electrode and a SAM of thiol of variable length confirms the exponential dependence of the rate constant on the chain length [
94], according to k
ET = k° exp[−βd]. In the case of SAMs, an additional parameter is the surface coverage, θ, after SAM formation. For a complete monolayer of SAM without any defects (θ = 1), the mechanism of ET is tunneling, as the monolayer shows blocking behavior. Therefore, the expression for the ET rate constant can be read as k
tunnel = k° exp[−βd], where k° is the rate constant for a bare electrode, β is the constant of electron transfer through tunneling, and d the thickness of the SAM. This is the case for chemisorption of long-chain thiols on a gold surface, which usually results in well-organized monolayers with minor defects due to effective van der Waals interactions along the carbon chains [
95]. Another mechanism is based on a membrane-like behavior of the SAM, so that redox species can permeate through the monolayer, and ET to/from the electrode takes place. This kinetic process is controlled by the partition coefficient, κ, between the solution and the membrane, the diffusion coefficient, D
f, in the membrane, and the kinetics of material transport at the film/solution interface with a rate constant, k
interface [
96]. In the presence of pinhole defects within the SAM, another ET pathway may be effective due to diffusion of the redox species through the defect sites to the electrode. This alternative transfer may operate in the case of short thiol chains which are expected to display less organized layers. All these ET phenomena may take place in parallel and an effective rate constant will reflect the relative contributions of the different pathways [
97] (
Figure 5). As an implication, for ET to proceed, a balance is needed between a sufficiently high ET rate and sufficient organization to be able to obtain a controlled SAM. This can be reasonably achieved by maintaining the length of thiols comprising around 6–8 carbons [
98].
The second critical issue is linked to the poor stability of the SAM, especially when the potential of the electrode is polarized to extreme values of potentials [
99]. The Au–S bond is found to be stable only in a small potential window i.e., −0.6 to +0.6 V vs. Ag/AgCl [
100]. Thiol-based SAMs are also sensitive to heating, and their thermal stability is limited to 400 K [
101]. Above this temperature, thiol molecules start to desorb in the form of disulfides, suggesting that the Au–S bond is weaker than the S–C bond of thiols [
102]. Some discrepancy, however, exists about the thermal stability of monolayers of thiol [
103]. SAMs formed by long-chain thiols appear to be more thermally stable due to their well-organized nature on the surface [
104].
As a consequence of the poor stability of the Au–S bond, a carbon–gold (Au–C) bond should be preferred [
105]. Reduction of aryl-based diazonium salts is a widely used method to prepare functionalized surfaces, not only on carbon, but also on gold electrodes [
106,
107,
108]. The interest in such modifications concerns the wide range of functional groups available that are associated to high stability over a large potential window. Formation of mixed monolayers by successive electrochemical reductions from a mixture of diazonium salts presents an added advantage towards surface functionalization [
109,
110]. The disadvantage is the difficulty to stop the reaction at the monolayer formation. Electrografting of aryl compounds on electrode surfaces is based on the formation and attachment of highly reactive aryl radicals. However, polymerization between two aryl radicals, and their chemical reaction with already adsorbed aryl molecules may compromise the quality of the monolayer. Possible control of the monolayer can be achieved by utilizing a so-called “protecting–deprotecting” approach, where a bulky group protects the functional group, avoiding both the formation of disordered multilayers and possible reaction of functional end groups with aryl radicals. Organization of the monolayer is typically controlled by the size of the protecting group [
111]. On the other hand, the chemical substituent groups attached to the benzene ring of diazonium salt can significantly affect the electrografting of these molecules on the electrode surface, first due to their size and second due to their nucleophilic/electrophilic nature. QCM measurement was used to probe the thickness of the electrodeposited aryl organic layer on an Au electrode, and suggested that the large size of the substituent and its steric hindrance typically led to the formation of thin layers [
112]. A recent study related to the kinetics of electrografting on gold surfaces also suggested that the presence of an electron-attracting group increases the rate of reaction of the aryl radical on the gold surface [
113], whereas the presence of an electron-donating group slows down the grafting process, thereby offering control over the possibility to form monolayers. It should be noted that a mixed layer of aromatic diazonium salt and thiol can be advantageously used, as demonstrated in the case of LAC on gold electrodes. A submonolayer of aryl groups was formed to minimize multilayer formation by aryl radical attack, while full electrode coverage was achieved by further thiol adsorption [
114].
Other tools to functionalize planar electrodes are noteworthy. Among additional immobilization strategies, a variety of amines can be covalently attached to the electrode surface through their electrooxidation onto the electrode [
115]. Immobilization via in vivo natural enzyme substrate can be used alternatively to significantly increase electrocatalytic activity relative to simple protein adsorption on the electrode. Bilirubin is the natural substrate for BODs, which is in vivo oxidized to biliverdin with the reduction of oxygen to water. BOD was immobilized on a pyrolytic graphite (PG) electrode prefunctionalized by bilirubin, and a twofold increase in electrocatalytic activity in terms of current was reported compared to a bare PG electrode [
116].
Infrared spectroscopy is a perfect tool to monitor an electrode functionalization and to judge the quality of the immobilization procedure regarding its impact on the protein structure. IR spectroelectrochemistry allows simultaneous monitoring of electrochemical signals and infrared spectra reflecting the secondary structure of the enzyme (Amide I and Amide II bands centered at ~1650 cm
−1 and ~1550 cm
−1, respectively). However, one important aspect when choosing an immobilization procedure is to avoid spectral interference between the immobilizing molecules and the enzymatic system to study. Since the electrode material used in spectroelectrochemical experiments is a metal (gold or silver), SAMs are the common platform used for enzyme immobilization. The spectral overlap between a SAM and a protein is usually negligible since the main signals arising from the alkyl chain are located in the high wavenumber region of the spectra (2800 cm
−1–3000 cm
−1). However, one has to pay attention to the chemical head group of the SAM, especially amine functions (N–H bending mode of primary amines from 1650–1580 cm
−1) and carboxylic functions (C=O stretching mode from 1760–1690 cm
−1). This issue is compounded when much more complex modifications of the electrode are required. This problem has been tackled in a very elegant manner for the immobilization of a membrane protein—the bacterial respiratory ubiquinol/cytochrome
bo3 (cyt
bo3) [
117]. In this study, a tethered bilayer lipid membrane (tBLM) was used to immobilize cyt
bo3. The commonly used lipid tether cholesteryl (2-(2-(2- mercaptoethoxy)ethoxy)ethyl)carbamate (CPEO3), which serves as an anchor for the lipid bilayer, contains a carbamate function that strongly overlaps with the protein signals. In this respect, the authors have successfully synthesized a novel molecule WK3SH (dihydrocholesteryl (2-(2-(2-ethoxy)ethoxy)ethanethiol), an IR transparent variant of CPEO3 lacking the carbamate function, thereby suppressing interferences with signals arising from the secondary structure of the enzyme. This effort allowed the challenging monitoring of a transmembrane proton gradient generated by cyt
bo3 catalytic activity.
3.3.2. Enzyme Engineering
Engineering on the enzymes can alternatively serve to tune the interaction between bare or functionalized electrodes. Although very elegant, this strategy has been less employed so far, mainly because it can affect both stability and activity of the protein before and after immobilization. However, unlike nonspecific adsorption where multipoint connections between enzymes and electrodes are possible, site-specific attachment can provide an orientational immobilization that will also dictate the distance between the enzyme active site and the electrode surface [
118,
119,
120]. The approach can thus maximize the rate of direct ET, with low distribution of ET rates. The basic approach is to use genetic engineering to introduce linkers on the functional proteins, then immobilize these protein molecules on the electrode via the linkers. The electrodes have to be chemically modified accordingly to specifically react with the labeled enzyme.
A classical site-specific immobilization method relies on the introduction of affinity tags like polyhistidine tags (His-tag), which are widely used in biochemistry for protein purification. His-tags have six sequential histidine residues that can chelate metals like Cu, Ni, or Co [
121], or can favor the electrostatic interactions towards hydrophilic surfaces [
122]. The issue here is to evaluate the effect of such site-specific immobilization of genetically engineered protein molecules compared to random immobilization on the interfacial ET efficiency. With His-tags being quite long linkers, the increased distance may impede the ET, or on the contrary may induce required flexibility of the immobilized enzymes. Oriented immobilization could be obtained via the formation of ternary metal chelate complexes between metalated nitriloacetic acid (NTA), such as a Cu-NTA functionality on the electrode, and His-tagged recombinant proteins. Balland et al. designed a short-length NTA-terminated alkane thiol to immobilize His-tagged LAC on a gold electrode through copper ligandation [
121]. Both N- and C-terminal His-tagged LACs were studied with the expectation of a more favorable orientation of LAC through the C-terminal labeling which is closer to the Cu T1. Although catalysis of O
2 reduction was reported in the presence of a redox mediator, no direct current could be obtained even in the most favorable C-terminal modification. This result strongly suggested that His-tag labeling at the C-terminal or N-terminal sequences in LAC does not allow Cu T1 to approach the electrode at a tunneling distance. In another study, deletion of a flexible 10-amino-acid sequence at the C-terminal end of a two-domain-type LAC was performed to expose the Cu T1. Labeling with a His-tag was additionally introduced in the mutated enzyme [
122]. The catalytic activity of the wild-type LAC was compared to the mutated one, once immobilized on Au electrodes modified by SAM displaying various chemical functionalities [
122]. Direct wiring of the enzymes was achieved and explained by the proximity of the C-terminal end to the Cu T1 in the LAC under study. Despite the fact that no significant difference in the surface concentration between adsorbed WT or mutants exists, direct electrochemical activity was reported to be much higher on mutated enzymes due to the oriented immobilization of genetically engineered enzymes. Li and collaborators introduced a cysteine-6-His-tag either at the C-terminal or N-terminal end of a LAC for an oriented immobilization on gold electrodes. The strategy was based on different distances to the Cu T1 or Cu T2/T3 between C-Ter and N-Ter. Based on the quantification of produced H
2O
2 during O
2 reduction, the authors proposed that a different orientation was obtained depending on the C-Ter or N-Ter grafting. It must be noticed, however, that no clear catalytic signals could be observed in both cases, underlining most probably a slow ET independently of the immobilization strategy [
123]. These three related examples illustrate the difficulty to rationalize the efficient immobilization of engineered enzymes for electrocatalysis.
Other peptides have potential binding properties for different types of surfaces. These peptides are expected to bind to surfaces by noncovalent interaction, and can exhibit high affinity and selectivity [
124]. They are mostly selected by the phage display peptide library [
125]. Few binding peptides display some affinity for material surfaces of interest for electrochemistry, such as for graphene [
126], carbon nanostructures [
127], and gold [
128,
129]. Some peptides are already used to anchor enzymes on different types of surfaces such as alkaline phosphatase on a gold surface [
129]. Currently, these peptides are not used to immobilize and orient redox enzymes on electrode surfaces, but they could provide a robust method to bind the enzyme at a different part of the protein. However, this binding method could also suffer from inhibition of the ET because of the length of the linker.
The publications just described above engineered only the N-terminal or C-terminal end. It should be interesting to have possibilities of mutation at other targeted parts of the enzyme, eventually closer to the active site, or at any locations on the enzyme surface in order to relate the position at which the enzyme is immobilized to the ET rate. As a recent illustration, cellobiose dehydrogenase from
Myriococcum thermophilum has been shown to be an ideal candidate for site-directed mutagenesis. Having no surface cysteine residues, this amino acid was introduced at specific surface locations in view of the oriented immobilization [
130]. “Thiol-ene” click chemistry between the thiol group available on the cysteine moieties and vinyl groups grafted on the electrode was successfully exploited for site-specific covalent linkage. The cysteine moieties were introduced on the dehydrogenase domain in such a way that two different orientations of the active site (the heme group of the flexible cytochrome domain) were ensured upon immobilization. An increase in electrocatalytic activity in terms of current for site-directed covalent immobilization was reported compared to physical adsorption, in a ratio ~7, thus indicating favorable enzyme loading on the electrode. Interestingly, a significant difference in current output was also observed between the two different grafting localizations, suggesting a control of the orientation of the enzyme. In the same way, a fungal laccase presenting a unique surface lysine residue close to the T1 Cu site was immobilized on planar electrodes [
131]. This strategy can be applied in all the cases where a unique surface residue can be genetically engineered. However, site-specific protein engineering is sometimes limited by the available sites on the protein surface and/or because such modifications might alter the function or structure of the protein. Homology modeling can also be used to design new mutants with enhanced catalytic activity and immobilization yield on a surface. This was the case in the work of Gao et al. who performed site-directed mutagenesis on formate dehydrogenase immobilized on nanoparticles [
132].
An attractive new strategy is the use of noncanonical amino acids (ncAAs) like propargyl-
l-lysine (PrK), which can be genetically introduced at desired locations on the enzyme [
133] (
Figure 6). Moreover, a unique alkyne chemical handle of ncAAs will ensure a covalent linkage of the mutated enzymes with the modified electrode via click chemistry [
133]. By varying the length and position of the linker on the
E. coli CueO, the distance of the enzyme’s electroactive site relative to the glassy carbon electrode was controlled [
134]. It was shown that (i) site-directed anchorage was more efficient for direct O
2 reduction than nonspecific immobilization; and (ii) labeling far from the active site was not favorable to high catalysis efficiency. However, it was also emphasized that the ET efficiency was not directly related to the distance between the active site and the electrode, suggesting that other pathways such as ET through the structure of the protein may be involved. In that particular study, one can wonder whether the flexibility of the linker could be a major factor governing the overall ET.
The effectiveness of oriented immobilization and electrochemical operational stability for these genetically modified enzymes on electrode surfaces certainly depends upon the immobilization strategy. In case of chelation between a histidine moiety on the modified protein and Cu–NTA ligands on the electrode, stability can be affected by dissociation of the Cu ion from the NTA-modified electrodes induced by competitive binding with the His-tag on the enzyme [
135]. In comparison, the operational stability of bioelectrodes prepared by covalent conjunction using click chemistry between cysteine-modified protein and vinyl groups of the electrode lasts up to a few days [
130].