This approach is quite simple and cheap since the enzyme is directly adsorbed on the chemically modified electrode. The idea to orientate the laccase via a “lock and key” or “plug-in-the socket” interaction with a substrate-like function at the electrode surface was first introduced by the group of Armstrong.438 They considered that the T1 Cu in the structure of
Tv-laccase (PDB ID 1KYA) was surrounded by a hydrophobic and electron-rich region, the “substrate-binding pocket”, where the oxidation of the substrate takes place. In their first studies, a wide variety of polycyclic aromatic molecules275,438 was covalently grafted on a “planar” PG electrodes. This led to more uniform and higher enzyme coverage of the electrode, as evidenced by epifluorescence microscopy. Importantly, the wave shape and the
114 onset of O2 electro-reduction were not affected by the chemical functions, thus attesting no
mediation nor change of the enzymatic mechanism. This result also underlines that the grafted chemical functions do not have to possess redox properties. However, the chemical modification, especially with anthracene and chrysene moieties, proved to be very good both for increasing the current density and improving the stability (Fig 28); leading to up to 4-fold increase in the current density. These results were attributed to the presence of specific hydrophobic and π-electron conductor recognition sites with the right length, angle and rigidity.
Figure 28. Catalytic cyclic voltammogramms of a laccase at a modified or unmodified PG electrode. Inserts show the grafted surface groups. (A) Pc-laccase at an electrode modified with anthracene groups. The black curves are the catalytic waves immediately after spotting on laccase solution and the red curves are the catalytic wave after cell solution was changed for fresh buffer. (B) Tv-laccase at an electrode modified with chrysene groups. Adapted with permission from [438] and [275]. Copyright 2007 and 2008, Royal Society of Chemistry.
In the following, most studies were performed with laccases from Pycnoporus cinnabarinus,275,438 Trametes versicolor,275,435,439,440 Trametes hirsuta,438, Cerrena
unicolor,441,442 but also with other enzymes with considerably different structure like the low-
115 The use of very smooth planar surfaces like monolayers obtained by protecting the electrode during the electrochemical grafting on polished glassy carbon electrodes444 or
closely packed SAMs on gold surface, 445,446 raised the question whether a good orientation is
only due to chemical modification or also to the roughness of the electrode. Very low DET was observed in these cases, although the electrode surface was decorated with anthracene or anthraquinone moieties. This result was explained by the fact that on a smooth surface, or on a well-organized monolayer, the chemical functions are less accessible. On the contrary, when a mixed (antracene-methanethiol/ethanethiol) SAM was formed on an electrochemically roughened gold surface, the grafted moieties became accessible to the laccase and the current doubled.445 A direct proof that isolated accessible molecules are needed for an Efficient
immobilization of laccase via supramolecular interaction was given by a SECM study of mixed SAMs on monocrystalline gold (111).446 Mixed thiolated veratric acid (tVA) /
mercaptopropionic acid (MPA) SAMs were built with different water contents, leading either to separated pure tVA or MPA phases or to a unique well-mixed phase. In the case of the pure phase, neighboring tVA moieties were closely interacting, thus forming clusters bigger than the enzyme hydrophobic substrate-binding pocket. This was directly linked to quasi-inexistent DET for adsorbed Tv-laccase on this SAM. On the contrary, tVA molecules were widely separated by MPA molecules in the well-mixed phase. Adsorbed Tv-laccase exhibited in this case a clear cathodic wave with j = 0.58 µA.cm-2 at 0.4 V vs. Ag/AgCl.446
This approach was further extended to different electrode materials and nanostructured architectures: carbon cloth,275 carbon nanotubes covalently modified with anthracene,
anthraquinone, naphthalene or related compounds at their ends or walls,424,439,441,442,447-450
CNTs covalently modified with bisphenyl groups,451 or CNTs non-covalently modified with
pyrene molecules bearing the desired chemical functions.440,452 Even on nanostructures, a
116 A major problem with CNTs is that the terminal « substrate-like » moieties are rich in aromatic electrons and can also bind to CNTs by π-stacking, thus reducing the amount of available anchoring groups for the attachment of the enzyme. Better availability of anchoring groups at the electrode surface can be induced by bi-functionality where at least one functional linker is not bound to CNT walls due to steric hindrance,436,452 as it has been done
with pyrene-bis anthraquinone,452 pyrene-bis naphtoquinone,436 or by functionalization of graphene oxide further immobilized by π-π interactions on MW-CNTs.348 Another possibility
is to use another hydrophobic moiety unable of π-stacking like adamantane.433 Interestingly,
although the binding energy of anthraquinone (evaluated by theoretical calculations) is almost twice higher than that of adamantane, catalytic currents for the reduction of O2 were much
higher with adamantane, reaching 2.2 mA.cm-2. This result, combined with QCM-D
experiments showed that more enzyme was immobilized, supporting the idea that more groups were accessible and allowed for enzyme binding and orientation.
The hydrophobic interactions between electrode and substrate-binding pocket was also exploited for SW-CNTs modified with a steroid biosurfactant bearing a large, rigid and planar hydrophobic moiety.453,454 This allowed very fast heterogeneous electron transfer rates
between T1 Cu site and SW-CNTs (3000 s-1). Hydrophobic polymers were also able to act as a conductive wire approached to the T1 via π-π stacking.455,456
However, a possible limitation of this approach is the lack of specificity of the hydrophobic interaction, since several hydrophobic regions co-exist at the surface of the enzyme, and not only close to the T1. This was evidenced in several cases by different experimental observations. Either a trailing edge was observed in the cyclic voltammograms275 or currents were consistently enhanced upon addition of a soluble
mediator, whatever the surface modification,444 consistent with the assumption that a unique
117
Sc-laccases on SW-CNT modified with the molecular tether PBSE was investigated via molecular dynamic. SW-CNTs (2.71 nm in diameter) were positioned near the hydrophobic substrate binding pocket of the laccases (position 1) and close to 2 other regions where adsorption was also likely (positions 2 and 3). In case of Tv-laccase, T1 Cu was situated resp. at 0.5, 2.1 and 3.0 nm from the surface in the considered positions 1, 2 and 3. Calculations demonstrated that adsorption was favored in region 1 over region 2 by 20-40 kcal.mol-1, but
by only 11-15 kcal.mol-1 in region 1 over region 3. For Sc-laccase, the distance between T1
Cu and the protein surface was resp. 0.7, 3.5 and 1.6 nm in the regions 1, 2 and 3. No preferential adsorption was shown between regions 1 and 3. This modeling study is a further indication that several positions of the enzyme at the electrode surface have approximately the same probability.457