Recent Developments of Nanostructured Electrodes for Bioelectrocatalysis of Dioxygen Reduction

The recent development of nanostructured electrodes for bioelectrocatalytic dioxygen reduction catalysed by two copper oxidoreductases, laccase and bilirubin oxidase, is reviewed. Carbon-based nanomaterials as carbon nanotubes or carbon nanoparticles are frequently used for electrode modification, whereas there are only few examples of biocathodes modified with metal or metal oxide nanoparticles. These nanomaterials are adsorbed on the electrode surface or embedded in multicomponent film. The nano-objects deposited act as electron shuttles between the enzyme and the electrode substrate providing favourable conditions for mediatorless bioelectrocatalysis.


Introduction
Although the first report on a dioxygen-glucose biofuel cell working at neutral pH was published in the early 60's [6], construction of electrodes suitable for this device became challenging research area in the late 90's.Similar to the conventional fuel cell dioxygen remains the oxidant in biofuel cells, because of its ubiquity, in particular its presence in the air and body fluids of mammals.Contrary to inorganic catalysts, enzymes allow for significant decrease of the dioxygen reduction overpotential also at neutral pH.This aspect is important if the biofuel cell is prepared to generate electricity in human body.Among the enzymes two types of copper oxidoreductases as high potential laccases and bilirubin oxidase [7][8][9] are superior, and electrodes modified by these proteins are the subject of almost all studies of prospective biocathodes.Although electrodes modified by high potential laccases allow for the larger decrease of the dioxygen reduction overpotential, bilirubin oxidase is more active at pH close to neutral as in physiological fluids [2,10].This property together with the fact that it retains its activity in the presence of chloride ions makes bilirubin oxidase modified electrode prospective biocathode working at physiological conditions without membrane separator.
However, the problem of its deactivation in the presence of other species such as urates [11] is still needed to be solved.
Both laccase and bilirubin oxidase contain two separate active sites embedded in polypeptide chains being close enough for electron transfer between them.Three-copper ion cluster is responsible for the binding of dioxygen and a separate copper ion responsible for electron transfer with the exterior substrate [22,23].Establishing direct electronic communication between the latter and the electrode surface becomes of central interest for enzyme-based mediatorless electrocatalysis.T1 center of laccase or bilirubin oxidase (Figure 1) is hidden in hydrophobic pocket of the protein, and it is accessible for electrons from electrode as it is for organic mediator.It has been shown that attachment of polyaromatic functionalities provides favourable conditions for efficient electron transfer to the acceptor site of the enzyme [1,[24][25][26].However, the catalytic current density obtained on these electrodes is limited by the low coverage of the electrode with the enzyme.The use of conductive nanomaterials like carbon nanotubes or various nanoparticles for biocathode modification provides an alternative and frequently used option.Due to the similar sizes of the flexible enzyme and the rigid nanoparticles (or other nanoobjects), the electron transfer distance can be decreased without bound to the trinuclear cluster (from [1]).
deterioration of enzyme activity [27,28].The increase of the electroactive surface after modification with nanomaterials is another important advantage.Reconstitution of the enzyme may also bring conductive nanoparticles close to the active site of the enzyme [29,30] possibly accelerating bioelectrocatalytic process.
Early papers from the Tarasevich group [36,37] are often cited as the first example of mediatorless catalysis with laccase adsorbed on carbon black.Indeed the high current density of dioxygen reduction reported on these materials may be connected with their specific nanosized features allowing for favourable orientation of the adsorbed enzyme.However, in the first few years of the 21st century the majority of research was oriented towards mediated bioelectrocatalysis following the concept of "wiring" the enzyme to osmium redox polymers [38][39][40][41].Later when the application of nanomaterials for electrode modification became increasingly popular, it was found that nanostructured electrodes further modified by copper oxidoreductases are suitable materials for mediatorless bioelectrocatalysis of dioxygen reduction [2,[42][43][44].Not only carbon nanoparticulate materials and carbon nanotubes but also films consisting of graphenes, metal or metal oxide nanoparticles were successfully applied for mediatorless dioxygen bioelectrocatalysis.This paper reviews the recent efforts to construct nanoparticulate biocathodes.

Electrodes Modified with Nanoparticulate Carbon Materials
Almost 30 years passed from the first observation of mediatorless bioelectrocatalysis at laccase adsorbed on carbon black [36,37] until a well-defined carbon-based nanomaterial was used for this purpose.The Kano group prepared a carbon aerogel (22 nm average pore size) poly(vinylidene fluoride) film on glassy carbon with adsorbed laccase or bilirubin oxidase [2].The use of a copper-oxidoreductasemodified carbon nanostructured material resulted in a significant overpotential decrease compared with Pt electrodes.
The electrocatalytic activity of these electrodes resulted in a dioxygen reduction current density of ca. 4 mA cm −2 as measured at rotating disc electrode (Figure 2).More importantly similar current densities were obtained in quiescent conditions when Toray carbon paper was used as support for the carbon aerogel [3] (Figure 3).This indicates that not only the structure and composition of the nanostructured film electrode but also the support material affect efficiency of the bioelectrocatalytic process.The extremely high current density (20 mA cm −2 ) was obtained in quiescent conditions with a polytetrafluoroethylene-Ketjen black composite deposited on carbon paper in contact with air [45].However, in these experiments another copper oxidoreductase-copper efflux oxidase-was used for modification of the air breathing cathode.Although very high current densities and remarkable stability can be obtained with this enzyme on mesoporous carbon supports [46], its dioxygen reduction overpotential is too large to be applied in a biofuel cell.
Another commercially available carbon black-Vulcan XC72R-embedded in a Nafion film with bilirubin oxidase provides a rather modest 0.05 mA cm −2 catalytic current determined by cyclic voltammetry at 0.003 V s −1 in quiescent O 2 -saturated solution [47].Gas-diffusion electrodes with immobilised bilirubin oxidase [48] or laccases [4] were prepared with the same carbon material hydrophobized with Teflon.With the first enzyme catalytic current density above 0.1 mA cm −2 was determined by cyclic voltammetry at 0.02 V s −1 and up to 0.35 mA cm −2 at 0.5 V versus Ag/AgCl from polarisation curve [48].Polarisation curves also show a three times increase of the current as compared with aqueous dioxygen supply.Even larger current densities and excellent stability was observed for a similar laccase-modified electrode with significant difference between tree and fungal laccases (Figure 4) [4].
Based on a similar principle, "floating," air diffusion biocathode prepared from hydrophobic carbon black modified with Trametes Hirsuta laccase exhibits a current density up to 0.5 mA cm −2 on a dioxygen saturated buffer solution (pH 4.5) [49].Importantly this electrode is reported to lose only 5% of its initial activity after 1 month of operation at low current density (0.02 mA cm −2 ) [49].
One of our groups proposed commercially available hydrophilic carbon nanoparticles functionalised with phenyl sulphonate groups (known as Emperor2000) as biocathode material [50].The film electrode consisting of these nanoparticles encapsulated into a hydrophilic sol-gel-processed silicate and further modified with adsorbed laccase exhibited mediatorless catalysis with ca.0.1 mA cm −2 current density obtained by slow scan (0.001 V s −1 ) cyclic voltammetry in quite conditions.Current density in a similar range was obtained with adsorbed bilirubin oxidase on the same nanoparticles embedded in hydrophobic silicate film functionalised with methyl [5] or imidazolium [51].It is worth noting that the addition of hydrophilic carbon nanoparticles to hydrophobic silicate-carbon microparticles composite material (carbon ceramic electrode) leads to an increase in the catalytic current density to 0.2 mA cm −2 as measured by cyclic voltammetry with scan rate 0.001 V s −1 in air-saturated solution [5] (Figure 5).
Scanning electrochemical microscopy studies revealed significant deaggregation of laccase [50] and bilirubin oxidase [5] by the presence of carbon micro-or nanoparticles in the electrode material (Figure 6).However some enzyme aggregates can be found by atomic force microscopy.
With functionalised silicate sub-microparticles the stable film is obtained due to electrostatic interactions between particles of opposite sign, and electronic conduction is secured due to the formation of percolation paths by the carbon nanoparticles (Figure 8).
By increase of the number of alternate immersion and withdrawal steps of the substrate into nanoparticles suspensions a larger amount of material is deposited (Figure 7).With adsorbed laccase or bilirubin oxidase these films exhibit mediatorless bioelectrocatalytic activity towards dioxygen reduction, and it is controlled by the number of immersion and withdrawal steps [12,52] (Figure 9).With adsorbed bilirubin oxidase a catalytic activity above 0.3 mA cm −2 (measured by cyclic voltammetry with scan rate 0.001 V s −1 in dioxygen saturated buffer) is attained.This is due to an increase of the electroactive surface where a larger amount of enzyme molecules can be adsorbed.Interestingly the bioelectrocatalytic activity of these electrodes is significantly larger than that prepared with functionalised sol-gel-processed silicate as a linker (electrode 1 in Figure 8) [52,54].This is perhaps due to the porous structure of the film obtained entirely from particles allowing for adsorption of the enzyme and relatively easy access of the substrate.However, chronoamperometric experiments show initial decrease of the current by ca.50% [52].

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The same concept was used to prepare nanoparticulate films composed of negatively charged carbon nanoparticles with phenylsulphonate functionalities and positively charged ones with sulphonamide-linked ammonium functionalities (Figure 10) [13].
Also these electrodes exhibit mediatorless bioelectrocatalysis of dioxygen reduction after adsorption of bilirubin oxidase and the magnitude of the catalytic current depends on the amount of material deposited on the ITO surface (Figure 11).
To summarize, the maximal catalytic current density obtained at film electrodes consisting of functionalised hydrophilic carbon nanoparticles by slow scan (0.001 V s −1 ) voltammetry under dioxygen saturation is in the range of 0.1-0.4mA cm −2 .
Another carbon-based material with nanosized poresmesoporous carbon was also used as support for laccasemodified biocathode [55].It exhibits catalytic current in the range of 0.1-0.3mA cm −2 as measured by slow scan (0.001 V s −1 ) voltammetry at pH 6.
Quite remarkable results were recently reported for bilirubin oxidase adsorbed on micro/macrocellular foams [56].These porous materials can be obtained in a somewhat controlled process, and their pore size ranges from tens of nanometers to tens of micrometers.With this material, the catalytic current density measured at 1000 rpm with scan rate 0.005 V s −1 in O 2 -saturated solution of pH7.2 achieves 2 mA cm −2 .

Electrodes Modified with Carbon Nanotubes and Graphenes
Not surprisingly carbon nanotubes are the most frequently used nanomaterials for biocathode modification.They can be used for building extended conducting networks with large effective surface area providing good electronic communication with the active site of the enzyme without decreasing its activity [8,9,[57][58][59][60].In addition, they can be easily functionalized through covalent bonding at the edges and defect sites or sidewalls of the carbon nanotube or noncovalently at the sidewalls [61,62].Chemical modification of the nanotubes sidewalls or its terminus is generally needed to control their dispersion and is crucial for the construction of devices [63][64][65].In biosensing applications, functionalization can provide biocompatibility and specific interactions with biological molecules in solution [66,67].
If mediators are employed to transfer electrons between the electrode and T1 center of oxygen by laccase or bilirubin oxidase, their leaching to the solution, and as a result a fast decrease of catalytic currents, as well as side reactions with the enzymes or other components of the biocathode or bioanode (in biofuel cells) are the adverse effects.This stimulates research on their immobilization, most frequently of ABTS 2− (2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonate), on carbon nanotubes or graphenes [15,16,65,[68][69][70][71][72][73].
Carbon-nanotubes-based biocathodes employing ABTS 2− as the mediator dissolved in solution were consedtruct from multiwalled carbon nanotubes (MWCNTs) functionalized with chitosan and laccase [74].MWCNTs-ionic liquid gel and laccase [75] or single-walled carbon nanotubes (SWCNTs) and bilirubin oxidase were encapsulated in silicate matrix [76].However these materials require the presence of a mediator in the oxygen-saturated catholyte.Therefore, membrane had to be employed to separate the glucose oxidase and SWCNT-modified anode from the catholyte [74,76,77].This is not necessary when ABTS 2− is immobilised on the electrode surface, for example, attached to the walls or ends of SWCNTs [14,65,68,78,79].This approach leads to the dioxygen reduction current density to 0.4 mA/cm 2 for electrodes covered with SWCNT-amine-ABTS and 0.2 mA/cm 2 for SWCNT-ABTS-side and Nafion layer containing laccase (Figure 12) [80].
Three ways of covalent modification of SWCNTs with ABTS 2− have been recently proposed (Figure 12).Their purity was evaluated by TGA (Table 1), because impurities or unintended functionalization may give rise to catalytic behavior [81].Moreover, TGA allows for the determination of the degree of nanotube modification expressed as the ratio of the number of moles of substituent to mole of carbon (Table 1) [14,[78][79][80].
For pristine SWCNTs, four main peaks should be present in the Raman spectrum: D, G, G and a low frequency peak (ω RBM < 300 cm −1 ), called radial breathing mode (RBM) (Figure 13).
RBM corresponds to the symmetric in-phase displacements of all carbon atoms in SWCNT in the radial direction and its frequency is related to the nanotube diameter [15].The multicomponent tangential G band is derived from the in-plane Raman-active mode in graphite.The RBM and multi-G-band are the first-order Raman features, which are characteristic only for SWCNTs.The last two main bands present in SWCNTs Raman spectrum are the disorderinduced D band (at ω D ∼1350 cm −1 ) and its overtone, the harmonic G band, at a frequency equal to ∼2 ω D .The dispersive D mode is observed when the symmetry of hexagonal sp 2 bonded lattice is destroyed, so it is related to the presence of the defects in the nanotube structure or amorphous carbon material in the tested sample.The intensities of all observed modes in Raman spectra can  change and the frequency of modes can be shifted due to chemical modification (Figure 13).The relative intensity ratios of Raman bands for pristine and modified SWCNTs also change.Commonly, the direct I D /I G intensity ratios are utilized to monitor the purity of the nanotubes and functionalization of the CNTs (Table 2), and I D and I G intensities are measured relative to the intensity of the two-phonon G mode, I G .The latter is decreased when uniformity of the sample decreases due to impurities or large distribution of nanotube sizes.Interestingly, the D peak is not observed to increase as a result of adsorption of, for example, hydronium ions or surfactants on the nanotube sidewalls, thus it allows for distinguishing covalent from noncovalent modification.
For nanotubes modified with azobenzene and anthraquinone, the frequency shifts of the observed bands were negligible; however the intensity of the D band increased Experiments were performed in dioxygen-saturated 0.1 mol dm −3 phosphate buffer (pH 4.8).Scan rate 0.001 V s −1 (from [12]).

nm
Figure 10: immersion and withdrawal steps alternatively into cationic and anionic carbon nanoparticles suspensions (from [13]).

Sample
Comparing the I D /I G ratio, which is 0.12 for pristine SWC-NTs, 0.26 for SWCNT-anthraquinone, 0.67 for SWCNTazobenzene, and 0.34 for SWCNT-ABTS (Table 2), we can find that the disorder is enhanced after modification.Additionally, grafting of the moieties to the nanotube sidewalls leads to changes of strength and frequency of RBM mode [15].Anthraquinone-derivatized SWCNTs produced a significant spectral shift of the radial breathing modes from 269 to 380 cm −1 .This is a strong indication of sidewall functionalization and disruption of the radial breathing motion due to the added functionality.The assessment of carbon nanotubes modification by Raman spectroscopy is extremely important when functionalities are not electroactive.This is the case of pyrene with hydrophilic functional groups, adsorbed on SWCNTs due to π-π interactions [21].After modification characteristic bands partially overlapping with the SWCNTs G-band and additional band at 1230 cm −1 from adsorbed pyrene can be seen.Additionally the ratio of D/G bands increases compared to that of pristine nanotubes confirming large extent of pyrene functionalization [21,84].
SWCNTs with covalently attached ABTS 2− moieties were used for preparation of biocathodes [14,79].The voltammogram of glassy carbon electrode modified with SWCNTs-ABTS and SWCNTs-ABTS-side and covered with lipid liquid crystalline cubic phase film containing laccase exhibits pair of peaks characteristic of mediator redox process [14,79] (Figure 14).In the presence of oxygen both electrodes show mediated bioelectrocatalysis with end modified SWCNTs more efficient (0.6 mA cm −2 compared to 0.4 mA cm −2 ) (Figure 14).These values are much higher than those obtained for similar electrodes with SWCNTs modified by adsorbed ABTS 2− (0.09 mA cm −2 ) or with the same mediator in solution (0.02 mA cm −2 ).The redox activity of the mediator also helps in the determination of the degree of SWNTs modification from the magnitude of cyclic voltammetry signal.For electrodes modified with MWCNTs encapsulated in silicate sol-gelprocessed film and then modified with adsorbed ABTS 2− less than 10% of the surface is covered by mediator molecules [68].These electrodes exhibit catalytic current density below 0.1 mA cm −2 as measured by slow scan (0.001 V s −1 ) voltammetry in dioxygen-saturated solution, ten times larger than in the absence of adsorbed mediator [68].Slightly larger current density was obtained with the same ABTS 2− functionalized carbon nanotubes stabilized with Nafion [65].Larger signal is observed from SWCNT-ABTS-side as compared to SWCNT-ABTS encapsulated in Nafion film together with laccase (Table 3).Also catalytic current density of this electrode is larger [79,80] (Table 3).Interestingly the catalytic efficiency per mediator molecule is significantly larger when mediator is attached to the end of carbon nanotube (Table 3).On the other hand, the total current density is larger when modification takes place at the sidewalls since the number of functional groups is larger.Modification of the electrodes with ABTS 2− functionalized SWCNTs allows to obtain catalytic current density up to 0.5 mA cm −2 and to decrease the onset potential of dioxygen reduction to 0.5 V versus Ag/AgCl making these biocathodes suitable for biofuel cells.Carbon nanotubes can be organized perpendicularly to the electrode surface, which allows utilization of their extended surface and takes advantage of the fast electron transfer along their main axis [85].Very recently vertically aligned MWCNTs were used for biocathode preparation [16].These electrodes were prepared by a CNT-transfer technique attaching the as-grown carbon nanotubes to ITO substrate with a glue prepared from MWCNTs and epoxy.ABTS 2− and laccase immobilization produce a not-veryefficient catalytic system with the current density not exceeding 0.1 mA cm −2 as measured by voltammetry with scan rate 0.001 V s −1 (Figure 15).Replacement of ABTS 2− with syringaldazine increases catalytic current eight times.However, the onset potential of the catalytic current is not as positive as in case of ABTS 2− due to the lower redox potential of syringaldazine.
The application of buckypaper-based electrodes prepared from MWCNTs and exhibiting apparent electrical conductivities up to 2500 S m −1 as biocathodes was recently reported [86].This material was further impregnated with ABTS 2− salt, Nafion, and bilirubin oxidase, and it allows obtaining catalytic current ca.0.2 mA cm −2 .
Remarkable stability of the enzyme was obtained with screen printed electrodes with MWCNTs, laccase, and ABTS 2− salt as ink component [87].Although the current density was not larger than 0.02 mA cm −2 , this way of nanoparticulate modification of cathode may become important in the future, because of the cost-effective manufacturing.
Another way of ABTS 2− immobilization on SWCNTsmodified electrode is based on their combination with layered double hydroxides to entrap and electrically connect laccase [88].The coating was based on electrostatic interactions between hydroxyl and carboxylic groups of chemically oxidized carbon nanotubes with the positively charged layered double hydroxides.Although the bioelectrocatalysis of dioxygen reduction with wired laccase was demonstrated, the current densities reported were relatively low [88].
Although few ways of carbon nanotube modification with mediator were proposed, the majority of research with this material is oriented towards mediatorless bioelectrocatalysis of dioxygen reduction.Non-functionalized MWC-NTs in silicate matrix with laccase exhibits current densities below 0.01 mA cm −2 measured by cyclic voltammetry with scan rate 0.001 V s −1 in dioxygen-saturated phosphate buffer pH 5.2 [69], whereas similar material impregnated with polyethylene glycol and bilirubin oxidase provides catalytic current above 0.1 mA cm −2 [89].A current density of ca.0.06 mA cm −2 , measured at scan rate 0.005 V s −1 , was obtained when the MWCNTs suspension in chloroform was casted on glassy carbon electrode and allowed to dry.In the second step, laccase was coadsorbed on this nanostructured surface and again left to dry [90].Non-functionalized SWCNTs with laccase provide ca.0.04 mA cm −2 measured by cyclic voltammetry with scan rate 0.001 V s −1 in dioxygensaturated phosphate buffer, pH 6 [91].Cross-linked bilirubin oxidase on similar electrodes provides 0.1 mA cm −2 at pH 7 [42].More importantly the catalytic current density increased more than five times when SWCNTs and crosslinked laccase were immobilized on a 7 μm diameter carbon fibre [92].This improvement is clearly due to specific geometry of the system, namely, enhanced mass transport, because of radial substrate diffusion.The replacement of laccase with bilirubin oxidase doubles catalytic efficiency of this micro-biocathode (Figure 16) [17].Modification of biocathode of similar geometry consisting of carbon nanotubes assembled on carbon nanofibers with osmium polymer and bilirubin oxidase results in the current density ca.1.6 mA cm −2 as measured with scan rate 0.005 V s −1 in phosphate saline buffer [93].This highly porous material in a form of microwire provides large contribution of radial diffusion not present at nanoparticulate flat film electrode.
Recently, a comparative study of bioelectrocatalytic dioxygen reduction on different carbon-based materials was published [18].Laccase adsorbed on graphite felt, porous carbon tubes, carbon nanofibers as well as SWCNTs and MWCNTs was characterised under comparable experimental conditions as half-cell electrodes using current densitycathode potential plots.Obviously, open circuit potential of all materials was significantly increased by laccase adsorption, indicating mediatorless catalysis.It was concluded that deterioration performance of these materials in the absence of mediator is due to electrode-material-dependent parameters, for example, oxygen diffusion within porous electrodes, enzyme adsorption or its orientation at the electrode surface.Among the investigated materials, MWCNTs show the best performance in terms of volume-normalized current density.However, the study also revealed that carbon nanotubes and porous carbon tubes exhibit dramatically lower current densities when normalized to BET surface area (Figure 17).This was ascribed to either materialdependent rate of direct electron transfer or limitations due to agglomeration of the nanotubes.Anyway, it also confirms that it is difficult to attain catalytic current density larger than 0.1 mA cm −2 (referred to as geometric area) with flat electrodes modified by nonfunctionalised carbon nanotubes and copper oxidoreductase.
The attempts to increase the efficiency of carbon nanotube biocathodes by preparing three-dimensional structures on the electrode surface have to be noted [19,73,94].In some experiment multilayered film composed of polylysine, MWCNTs, and laccase was prepared [19,94].A controlled amount of nanotubes and enzyme was immobilised using layer-by-layer technique (Figure 18(A)).Efficient bioelectrocatalysis with current density over 0.5 mA cm −2 (Figure 18(B)) allowed for the use of this biocathode in a glucose-O 2 biofuel cell [19] and as a self powered biosensor [94].
A three-dimensional carbon nanotube-hydroxyapatite nanocomposite film was also used as a biocathode material; however, rather low catalytic efficiency was achieved [73].
Very impressive catalytic efficiency was achieved with biocathodes made of directly grown SWCNTs [20].Laccase was immobilized on carbon nanotube forest by liquidinduced shrinkage (Figure 19), and a catalytic current density up to 4 mA cm −2 was obtained.
There are many reports exploring modification of carbon nanotubes to prevent their aggregation and promote direct electron transport to immobilised copper oxidoreductase and therefore mediatorless bioelectrocatalysis.SWCNTs non-covalently functionalized with 1-pyrene derivatives and immobilized in hydrophilic (obtained from tetramethoxysilane) and hydrophobic (obtained from methyltrimethoxysilane) silicate matrix were used to enhance direct electron transfer to enzyme laccase [69] and bilirubin oxidase [21].The enzymes were either adsorbed from the solution or coimmobilized with the nanotubes.In the former case the charge of the functionalized nanotubes was decisive for the efficiency of bioelectrocatalytic reduction of oxygen.It was also shown that the functional group of adsorbed pyrene affects the magnitude of catalytic current, and the use of 1-pyrene sulfonate is most favourable for bilirubin oxidase modified electrode (Figure 20).The importance of electrode functional groups and superiority of sulfonate was also found in fundamental studies of bioelectrocatalytic dioxygen reduction on gold [26,95].Interestingly for laccase-modified electrodes four times larger catalytic current is observed for vertically aligned MWCNTs modified with 1-pyrene sulfonate [16] indicating importance of film geometry.These electrodes prepared were used as cathode zinc-dioxygen batteries [16,21].SWCNTs with attached nonelectroactive aromatic groups either on the side or at the ends of the carbon nanotubes (Figures 21 and 22) were explored for biocathode modification [31].Aryl functionalized nanotubes were expected to enter the hydrophobic pocket of the T1 centre of copper oxidoreductases.The approach of Sosna et al. [26] allows binding anthracene and anthraquinone to the electrode by deprotecting amine linkers organized in a monolayer; however, the population of laccase molecules bound this way to the monolayer modified electrode is very small.Nanostructuring the electrode with SWCNTs covalently modified with anthraquinone or anthracene leads to a visible increase of the oxygen catalytic current since it allows much more laccase to be hydrophobically bound to the electrode surface.The 2D type of binding is transformed this way into a 3D assembly connected electrically [31].Anthraquinonemodified SWCNTs were obtained from acid chloride functionalized nanotubes (Figure 21).This functionality reacts with amine substituted anthraquinone.

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Synthesis of SWCNTs arylated on side walls was carried out by free radical reaction.The first step involves generation of diazonium compounds from aromatic amines.This intermediate was thermally decomposed in the presence of carbon nanotubes under permanent sonication at elevated temperature (60-65 • C) (Figure 20).Arylated SWCNTs were deposited on glassy carbon surface in three ways (Figure 23).This affects catalytic current density: the highest is observed for the electrodes first modified with a layer of arylated SWCNTs casted on the GC surface and then covered with the matrix, for example Nafion, lecithin, chitosan, or lipid liquid crystalline cubic phase, containing laccase and the same arylated SWCNTs [14,31,78,79].
The type of functional group also affects catalytic current density (Table 4), and its largest value was obtained with terphenyl functionalized SWCNTs.On the other hand, for all these electrodes showing DET, the onset potential is close to the formal potential of laccase T1 site.(Figure 24) [32].These electrodes were also successfully applied as cathodes in a hybrid biofuel cell with a Zn anode [32].
Wrapping SWCNTs with single-stranded DNA was shown to decrease the shear stress between the enzymes and SWCNTs [96,97].
Enzymatic functionalization of nanotubes as well as graphene nanosheets would allow appropriate orientation of the enzyme molecule and efficient electrical connection.The use of these materials provides also greater surface area for anchoring enzymes and improving kinetics of the enzyme [98].Indeed, covalent linking of multicopper oxidases to well dispersed conductive carbon nanotubes represent alternative strategy to facilitate direct electron transfer and efficient bioelectrocatalytic oxygen reduction [33,34,70,[99][100][101].
Irreversible adsorption of pyrene moieties on carbon nanotubes and their functionalization with copper oxidoreductase was explored [33,99].Linking laccase or bilirubin oxidase to MWCNTs via the molecular tethering reagent 1pyrenebutanoic acid, succinimidyl ester, results in a catalytic current efficiency in the range of 0.3-0.4mAcm −2 [99].Similar way of laccase bonding based on reaction of 1-aminopyrene with glutaraldehyde was proposed (Figure 25) [33].Although the catalytic efficiency of this system is not very impressive, the authors report that after one week the electrode retains 85% of its initial activity, which is much better than for analogous electrode with adsorbed enzyme.
Schubert et al. [34] immobilized bilirubin oxidase on gold by means of thiol-modified MWCNTs fixed on the gold electrode.The enzyme was covalently bonded to the other end of carbon nanotube (Figure 26).The direct electron transfer process led to remarkably high bioelectrocatalytic oxygen reduction current (50 μA cm −2 measured by cyclic voltammetry at 0.01 V s −1 in air-saturated buffer, pH 7) and rate constant of enzymatic reaction equal to 80-100 s −1 .In an earlier study the selection of interlayer compound for direct electronic communication of the enzyme with the electrode was emphasized.This indicates the importance of the linkage for electrodes stability, and indeed these electrodes retain 50% of their activity after 16 days of storage.
Not surprisingly several examples of the application of graphene sheets for biocathode construction have been reported recently [70,[102][103][104].Biocathode consists of a gold substrate on which bilirubin oxidase, graphene were coimmobilized using silica sol and gel matrix [102].The catalytic efficiency of this electrode in the presence, ABTS 2− salt in solution was not very high; however, authors emphasize the importance of graphene in the enhancement of stability of the constructed biofuel cell.Some improvement was seen with similar electrode based on a polypyrrole matrix [70].

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Electrode system based on graphene integrated with laccase and ABTS 2− was developed for detection of the extracellular oxygen released from human erythrocytes [103].Similar to the case of carbon-nanotubes-modified electrodes [65,68] strong π-π interactions were employed for mediator immobilization on the graphene sheets, whereas immobilization of laccase involves electrostatic interactions [103].This electrode exhibits mediated electrocatalysis, and the use of graphene increases electrocatalytic activity of the glassy carbon electrode 5 times.In the absence of mediator no bioelectrocatalysis was observed.The use of graphene nanoplatelets for laccase-modified biocathode was reported but this electrode shows only mediated catalysis with ABTS 2− salt in solution [104].
At the moment no advantage of the replacement of carbon nanotubes with graphene for biocathode construction is seen.Interestingly current research has also not shown any clear advantages of this material in the case of electrochemical biosensors [59].

Electrodes Modified with Metal and Metal Oxide Nanoparticles
There are not so many examples of metallic nanostructured films used for biocathode preparation in comparison with carbon-based materials.In the case of gold this may be due to unfavourable orientation of copper oxidoreductase on this material [105].
Nanoporous gold electrodes with a pore size in the range of 10-100 nm with adsorbed laccase exhibit enzyme direct voltammetry and modest bioelectrocatalytic activity.Catalytic current equal to 0.03 mA cm −2 was recorded by cyclic voltammetry with scan rate 0.1 V s −1 in air-saturated buffer solution (pH 4.4).Further studies of this material revealed that a larger pore size promotes adsorption of a larger amount of the enzyme.Interestingly from the point of view of stability, physical adsorption of laccase was found to be a superior immobilisation method, better than covalent binding or electrostatic attraction.
The study of glassy carbon electrode modified with citrate-stabilised Au nanoparticles (average diameter 50 nm) and laccase also reveals low (0.01 mA cm −2 ) catalytic current density determined by slow voltammetry (at 0.001 V s −1 ).However, contrary to other studies involving nanostructured metal electrodes the redox signal of the enzyme can be seen.This study also shows that electron transfer rate between T1 site of laccase and gold is similar to that obtained at carbon materials.Interestingly the authors showed that the presence of Au nanoparticles decreases laccase activity.Three-dimensional gold nanoparticulate electrodes can be also obtained by casting 15 nm Au nanoparticles suspension on polycrystalline Au electrode [106].This procedure is followed by modification of the resulted film with 1-propanethiol and adsorption of bilirubin oxidase.On such electrode a catalytic current density 0.5 mA cm −2 (as measured by cyclic voltammetry at 0.01 V s −1 ) is reported in quiescent O 2 -saturated solution at pH 7. The catalytic current is also a function of the functional group of the adsorbed thiol (COOH, NH 2 , OH, and CH 3 ) with carboxylate group having superior effect.Activity of these electrodes is perhaps the largest observed for metallic support, and they exhibit also that remarkable stability and a further substantial increase of the current can be achieved with the application of carbon paper as support.
Mediatorless bioelectrocatalysis of dioxygen reduction was recently reported on Au nanoparticles immobilised in a film composed of partially sulfonated (3-mercaptopropyl)- trimethoxysilane sol-gel-processed silicate, chitosan, and laccase [107].This material immobilised on carbon felt provides extremely high current density up to 4 mA cm −2 .This biocathode was applied in biofuel cell powered by alcoholic beverages.
Sophisticated approach employing Pt nanoparticles (2 nm) for biocathode construction was recently reported by Willner group [35,108].It is based on the modification of bilirubin oxidase by a polymerizable thioaniline monomer (Figure 27).This procedure affects enzyme activity only to very small extent and allows for preparation of a bioelectrocatalytic polymeric film at thioaniline-modified Au or Pt black surfaces.This is done electrochemically in the presence of thioaniline-appended bilirubin oxidase and thioanilinecapped Pt nanoparticles.
Such modification allows to obtain catalytic current density in the range of 0.5-0.8mA cm −2 .Most importantly this value is ca. 10 times higher than that obtained in electropolymerised film with encapsulated non functionalised enzyme (Figure 28).
According to the authors the enzyme functionalisation establishes better electrical contact between the Pt nanoparticles and partially unfolded enzyme or enables cooperative action of both species [35].This paper shows one of the possible research paths to increase the efficiency of the bioelectrocatalytic process.
Recently nanoparticulate electrodes consisting of metal oxides were tested for dioxygen reduction bioelectrocatalysis with adsorbed bilirubin oxidase [109].The modest catalytic effect was observed for nanocrystalline hematite (α-Fe 2 O 3 ) due to proline residue binding affinity.Generation of photocurrent is also possible in the presence of dioxygen under illumination conditions.This effect is not seen with nanocrystalline TiO 2 or WO 3 [109].O 2 reduction electrocatalysis with a current density above 0.1 mA cm −2 as measured by cyclic voltammetry at 0.001 V s −1 is also seen with a film electrode prepared from 10 nm diameter ITO nanoparticles [110].Interestingly, 0.015 mA cm −2 current is seen on bare ITO without any surface modification.This is perhaps due to the inherent nanostructure of ITO film on glass which can be seen under scanning electron microscopy.Additionally ITO substrate is more favourable materials than α-Fe 2 O 3 from the point of view of overpotential decrease.

Conclusions
In the last few years we have observed a great research effort to develop nanoparticulate biocathodes.This is due to the significant development of nanotechnology and the requirements of elimination of mediators from biofuel cells and electrochemical biosensors.Many systems based on electrodes modified with nanoobjects and biocatalysts of dioxygen reduction exhibit direct electron transfer and therefore mediatorless bioelectrocatalysis.
One may argue that, although the electron shuttle molecule is eliminated, a new, larger element (nanoparticle, nanotube) is introduced to the system.However, in the latter case a different mechanism of electron transfer is operative, based on electronic conductivity and not on the redox processes of the mediator.The notable advantage of such mechanism is that it does not affect the stability of the nanoobjects to any significant degree.On the other hand, the possibility of aggregation of the nanoparticles and restructuring of the film has to be taken into account.
Many nanoparticulate film biocathodes exhibit mediatorless bioelectrocatalysis and current densities competitive with those achieved using osmium polymers.There are few examples of particularly high current densities (up to 4 mA cm −2 in quiescent conditions) [17,20,107].This may be achieved by means of specific film structure [20] or specific geometry of the electrode, for example, using microwire [17] or carbon felt support [107].The majority of nanoparticulate biocathodes exhibit current densities in the range of 0.05 to 0.5 mA cm −2 , and we believe that there is still room for improvement.The development of new electrode preparation procedures resulting in novel architectures represents important outcome of the research on biocathodes.
Unfortunately, in most cases a significant decrease of the current is seen during the first hour of biocathode operation.This is due to not only decrease of the copper oxidoreductase stability and/or their weak bonding to the electrodes but also restricted access of dioxygen to the active sites located in the film pores.This difficulty can be at least partially overcome by the preparation of films with larger pores [56].Another approach is to design electrodes which provide direct access of gaseous dioxygen to the active sites of the biocathode.Therefore, very recently we could witness the development of "air breathing," gas-diffusion biocathodes [4,45,48,49,111].

Figure 1 :
Figure 1: Three-dimensional representation of the copper binding sites of bilirubin oxidase from Myrothecium verrucaria.The copper centers of T2/T3 type are represented by brown spheres, whereas the T1 copper is represented by blue sphere.Red sphere represents O 2 bound to the trinuclear cluster (from [1]).

2 E 1 E− 2 )Figure 4 :
Figure 4: (a) Current-voltage behaviour of fungal (squares, triangles) and tree (circles) laccase-based gas diffusion cathodes.Influence of oxygen on the polarisation curve obtained for fungal laccase in air (triangles) and oxygen (squares) stream conditions.(b) Tafel plots obtained from polarisation data for fungal (squares, triangles) and tree (circles) laccase.(c) The difference between potentials obtained in oxygen and air as a function of current density (from [4]).

EHT = 1 .Figure 7 :
Figure 7: Scanning electron microscopy images of ITO electrode coated by one (a) and three (b) immersion and withdrawal steps to cationic silicate particles and carbon nanoparticles suspension alternatively (from [12]).

Figure 17 :Figure 18 :
Figure 17: Current density-cathode potential plots for the electrodes with laccase adsorbed on the following materials: graphite felt (a), porous carbon tubes (b), single-walled carbon nanotubes (c), multiwalled carbon nanotubes (d), and carbon nanofibers (e).(A) Comparison of cathode performance with a current density normalized to volume.(B) Comparison of cathode performance with a current density normalized to BET surface area.Reproduced with permission (from [18]).

Figure 19 :− 2 )Figure 20 :Figure 21 :Figure 22 :Figure 23 :
Figure 19: Schematic diagram of enzyme entrapment inside a carbon nanotube forest by liquid-induced shrinkage.The photograph in the right upper corner shows a free-standing enzyme carbon nanotube forest ensemble film that can be manipulated with tweezers (from [20]).

Figure 27 :
Figure 27: (a) Modification of bilirubin oxidase with polymerizable thioaniline functionality.(b) Synthesis of the 3D oligoaniline crosslinked Pt nanoparticles/bilirubin oxidase composite by electropolymerisation of the thioaniline-modified Pt nanoparticles and thioaniline modified bilirubin oxidase on the thioaniline monolayer functionalised Au electrode.(c) The same process as (b) on Pt-film-coated Au electrode (from [35]).