Michael Holzinger
email: michael.holzinger@univ-grenoble-alpes

Carbon based nanostructured bio-assemblies for bioelectrochemical applications

Keywords: Carbon nanotubes, Graphene, Biosensors, Biofuel cells, Functionalization, Immobilization of biomolecules, Enzyme wiring

Carbon nanomaterial gained high interest for bioelectrochemical applications like biosensors and biofuel cells related to excellent conductivities, well established (bio) functionalization methods, and high surface area to immobilize a high number of biomolecules preserving accessibility for its substrate (analyte or fuel). Beside the huge varieties of different nanosized carbon structures, the principal materials used in this field are carbon nanotubes and graphene. However, within these carbon allotropes, there are tremendous differences in physical and chemical properties related to different production methods which affects processabilities and therefore, the appropriateness for an envisioned application. Here, the historical evolution of these carbon nanomaterials is summarized with focus on the different properties of carbon nanotubes and graphene materials. For bioelectrical applications, some of these carbon materials are more beneficial than others in terms of immobilization and wiring of bioreceptor units and biocatalysts. The chemical composition and specific domains of the biomolecules can establish efficient interactions with the carbon materials and can be improved with targeted functionalization strategies. The aim of this review is to provide a pedagogic summary of the different properties of carbon nanotubes and graphene materials from a chemical point of view citing most relevant reviews and discussing original strategies to form operational bioassemblies for analysis and biological energy conversion.

Introduction

Nanostructured carbon materials are known since many decades, but the scientific community did not address much attention until the fully characterization of the molecular C 60 soccer ball by [START_REF] Kroto | C 60: Buckminsterfullerene[END_REF] [START_REF] Kroto | C 60: Buckminsterfullerene[END_REF]. Few years later in 1991, Sumio Ijima reported the formation of "Helical microtubules of graphitic carbon" using arc-discharge [START_REF] Iijima | Helical microtubules of graphitic carbon[END_REF]. Even when it is not the first description of multiwallled carbon nanotubes (MWCNTs) [START_REF] Radushkevich | O strukture ugleroda, obrazujucegosja pri termiceskom razlozenii okisi ugleroda na zeleznom kontakte (About the structure of carbon formed by thermal decomposition of carbon monoxide on iron substrate)[END_REF][START_REF] Monthioux | Who should be given the credit for the discovery of carbon nanotubes?[END_REF], it became the standard reference within the topic of carbon nanotube research. In fact, the simultaneous development of different synthesis methods in 1993 reported by Ijima [START_REF] Iijima | Single-shell carbon nanotubes of 1-nm diameter[END_REF] and Bethune [START_REF] Bethune | Cobaltcatalyzed growth of carbon nanotubes with single-atomic-layer walls[END_REF] to produce single-walled carbon nanotubes (SWCNTs) conducted to an avalanche of research efforts focusing this material. SWCNTs have exceptional electronic and mechanic properties. Such seamlessly rolled-up graphene sheets provide semiconductor or metallic conductivity depending on the roll-up vector which determines the diameter and position of the hexagons in the tubes [START_REF] Odom | Atomic structure and electronic properties of single-walled carbon nanotubes[END_REF]. The induced bending of sp 2 carbon around the tube axis provoke further a real one-dimensional conductivity at room temperature. These firstly observed phenomena fascinated the scientific community and SWCNTs were rapidly envisioned for high sophisticated electronic application [START_REF] Mceuen | Single-walled carbon nanotube electronics[END_REF]. SWCNTs further provide extremely high tensile strengths and young's moduli [START_REF] Liu | Mechanical properties of single-walled carbon nanotube bundles as bulk materials[END_REF][START_REF] Guo | Mechanical properties of single-walled carbon nanotubes based on higher order Cauchy-Born rule[END_REF] and were even considered for utopic applications like cables for space elevators [START_REF] Edwards | DESIGN AND DEPLOYMENT OF A SPACE ELEVATOR[END_REF]. MWCNTs are to some extend less performing in terms of (metallic) conductivity [START_REF] Ebbesen | Electrical conductivity of individual carbon nanotubes[END_REF] or tensile strengths but became the privileged material since the outer wall contain in general oxygenated defect functions which improve processability to form homogeneous conductive composites. [START_REF] Xu | Ultra light conductive carbon-nanotubepolymer composite[END_REF]. These exceptional properties of carbon nanotubes (CNTs) and applications are reviewed in many articles [START_REF] Popov | Carbon nanotubes: properties and application[END_REF][START_REF] Ebbesen | Carbon Nanotubes: Preparation and Properties[END_REF][START_REF] Dai | Carbon Nanotubes: Synthesis, Integration, and Properties[END_REF][START_REF] Coleman | Small but strong: A review of the mechanical properties of carbon nanotube-polymer composites[END_REF][START_REF] Buongiorno Nardelli | Mechanical properties, defects and electronic behavior of carbon nanotubes[END_REF][START_REF] Ajayan | Topics in Applied Physics[END_REF]. One issue in carbon nanotube research was that single-walled, double walled, thin multiwalled and thick MWCNTs provide different properties when synthesized by arc discharge, laser ablation, or chemical vapour deposition (CVD) under different conditions (e.g. catalyst, temperature, carbon source) which made the comparison of the various studies difficult [START_REF] Rahman | An Overview of the Recent Progress in the Synthesis and Applications of Carbon Nanotubes[END_REF][START_REF] Dresselhaus | Carbon Nanotubes: Synthesis, Structure, Properties, and Applications[END_REF]. A great step forward was the development of processes for CNT mass-production and commercialization with reasonable costs [22] which enabled the availability of identic CNT samples for the scientific community.

The interest in carbon nanotube research clearly decreased with the first isolation and characterization of monolayer graphene by A. Geim and K; Novoselov in 2004 [START_REF] Novoselov | Electric Field Effect in Atomically Thin Carbon Films[END_REF]. Similar to CNTs, graphene and related 2D carbon materials have been studied since several decades. H. P. Boehm firstly described the synthesis of monolayer flakes out of reduced graphite oxide in 1962 [START_REF] Boehm | Proceedings of the Fifth Conference on Carbon[END_REF] and used the term "graphene". The process for graphene oxide synthesis was developed by Hummers et al. [START_REF] Hummers | Preparation of Graphitic Oxide[END_REF] few years before and remained little noticed until the work of Geim and Novoselov. As for CNTs, there are also different types of graphene like monolayer graphene, bulk (few layer) graphene, graphene oxide, or reduced graphene oxide which are obtained by various methods [START_REF] Wick | Classification framework for graphene-based materials[END_REF]. Monolayer graphene can be produced by the scotch tape technique exfoliating one graphene layer from highly oriented pyrolytic graphite (HOPG) which Geim and Novoselov used for their first studies. The disadvantage is that only few graphene layers can be isolated and most of the deposited material are debris from HOPG. Almost defect free monolayer graphene can be obtained via CVD synthesis on copper foils [START_REF] Mattevi | A review of chemical vapour deposition of graphene on copper[END_REF]. The disadvantage here is that this graphene has to be transferred to other substrates before its use. In most cases, bulk graphene or related materials are obtained via top-down production methods from graphite via mechanical [START_REF] Yi | A review on mechanical exfoliation for the scalable production of graphene[END_REF] or chemical exfoliation [START_REF] Parvez | Exfoliation of graphene via wet chemical routes[END_REF]. Bulk graphene materials are quite easily to prepare with standard laboratory equipment out of graphite. The issue is, as is was for the beginning of carbon nanotube research, that research teams produces its own graphene, each with their own protocol, forming graphene samples with different properties which impede comparison of the results within the different topics [START_REF] Ye | Graphene at Fifteen[END_REF][START_REF] Cai | Methods of graphite exfoliation[END_REF][START_REF] Randviir | A decade of graphene research: production, applications and outlook[END_REF][START_REF] Tiwari | Graphene research and their outputs: Status and prospect[END_REF].

Beside CNTs and graphene materials, there exist further nanosized carbon allotropes such as carbon nanohorns [START_REF] Karousis | Structure, Properties, Functionalization, and Applications of Carbon Nanohorns[END_REF], carbon quantum dot [START_REF] Lim | Carbon quantum dots and their applications[END_REF], helical or coiled CNTs [START_REF] Hanus | Synthesis, Characterisation and Applications of Coiled Carbon Nanotubes[END_REF], fishbone-type carbon nanofibers [START_REF] Sebastián | On the importance of the structure in the electrical conductivity of fishbone carbon nanofibers[END_REF], nanodiamonds [START_REF] Nunn | Nanodiamond: A high impact nanomaterial[END_REF],or carbon (graphene) nanoribbons [START_REF] Dutta | Novel properties of graphene nanoribbons: a review[END_REF] (Fig. 1). All these materials are extensively studied for various applications like reinforced conductive composites, energy storage and harvesting, biomedical applications, or (bio)analysis [START_REF] Gogotsi | Not just graphene: The wonderful world of carbon and related nanomaterials[END_REF][START_REF] Loh | Clinical Applications of Carbon Nanomaterials in Diagnostics and Therapy[END_REF][START_REF] Liu | Tailoring the Structure of Carbon Nanomaterials toward High-End Energy Applications[END_REF] but are little represented in the field of bioelectrochemistry. For bioelectrochemical applications, CNT and bulk graphene materials are mostly used since simple deposits form highly porous extensions of a working electrode enabling improved interaction and immobilization of biomolecules. Further advantages of graphene materials and CNTs is that both can be shaped into free standing macroscopic electrodes while keeping enhanced porosities and conductivity [START_REF] Cong | Graphene-based macroscopic assemblies and architectures: an emerging material system[END_REF][START_REF] Liu | Macroscopic shaping of carbon nanotubes with high specific surface area and full accessibility[END_REF]. The technological progresses of these carbon nanomaterials for (bio)electrochemical applications are nicely reviewed by Maduraiveeran et al. [45] and Kour et al. [START_REF] Kour | Review-Recent Advances in Carbon Nanomaterials as Electrochemical Biosensors[END_REF] . Bioelectrochemical applications principally concern the fields of biosensors and biofuel cells. Biosensors are defined by its receptor unit which is an entity of biological origin like enzymes, DNA strands, antibodies, antigens, or related proteins. Biomimetic models like aptamers, molecular imprinted polymers, or synthetic peptides are nowadays well accepted as sensing elements in the biosensor field [START_REF] Campuzano | Affinity-Based Wearable Electrochemical Biosensors: Natural versus Biomimetic Receptors[END_REF]. Biofuel cells operate as classic fuel cells where the catalyst is an enzyme. For this technology, the challenge is not only to immobilize the biomolecule but also to establish electron transfers from and to the electrode material which consists nowadays of carbon nanomaterials. There are many review articles about carbon nanomaterials for biomedical applications [145-152] where many different techniques and for different analytes are discussed.

In this review, more the fundamental aspects of the beneficial properties of carbon nanomaterials in bioelectrochemistry are focused. Specific interaction of biomolecules with such nanostructures and efficient immobilization techniques are discussed with some original examples for promising biosensor and biofuel cell concepts.

Appropriateness of carbon nanomaterials in bioelectrochemistry

Conductive carbon is intensively used in electrochemical applications in form of glassy carbon, pencil graphite, HOPG. Carbon is electrochemically silent in a wide potential range which means that any electrochemical redox processes can be recorded without interferences of the electrode material. This potential range covers further all bioelectrochemical processes and is therefore a standard material in electrochemical biosensors and biofuel cells [START_REF] Mccreery | Advanced Carbon Electrode Materials for Molecular Electrochemistry[END_REF]. The nanosized sp 2 allotropes of carbon behave electrochemically almost the same as macroscopic carbon electrodes [START_REF] Pumera | The Electrochemistry of Carbon Nanotubes: Fundamentals and Applications[END_REF] but the formation of porous structures provokes an extension of the electrode with drastically enhanced surface area. Interestingly, there are little quantitative studies about the effective enhancement of the electroactive surface using nanocarbon materials [START_REF] Mosch | Specific Surface versus Electrochemically Active Area of the Carbon/Polypyrrole Capacitor: Correlation of Ion Dynamics Studied by an Electrochemical Quartz Crystal Microbalance with BET Surface[END_REF]. One reason might be the innumerable amount of different nanocarbons coupled with many different preparation methods such as drop casting, spin coating, screen printing, use of binders, or formation of composites. Graphenes and CNTs can also be shaped into free standing electrodes. The most famous examples are buckypapers which are generally formed by filtration of CNT or graphene dispersions [START_REF] Gross | Bioelectrochemistry[END_REF]. Other macroscopic shapes like fibres or pellets can also be formed with these carbon materials [START_REF] Holzinger | Dekker Encyclopedia of Nanoscience and Nanotechnology[END_REF][START_REF] Cheng | Graphene fiber: a new material platform for unique applications[END_REF]. Independent on processing or final shape of nanocarbon electrodes, as long as the nanosized structure is available for biomolecules, privileged interaction can be established related to their similar nanosized dimensions [START_REF] Pandey | Carbon Nanotubes, Graphene, and Carbon Dots as Electrochemical Biosensing Composites[END_REF][START_REF] Brun | Functional Electrodes for Enzymatic and Microbial Electrochemical Systems[END_REF][START_REF] Campuzano | Carbon Dots and Graphene Quantum Dots in Electrochemical Biosensing[END_REF].

Biofunctionalization of carbon nanostructures

One great challenge in bioelectrochemistry is the immobilization of biological entities on electrode surfaces without altering their biological activity. Related to vast diversity of biomolecules, targeted immobilization methods, tailored for each type of compound, categorized in proteins (enzymes, immune systems) and nucleotides (DNA, RNA, aptamers), were developed [START_REF] Cosnier | Encyclopedia of Applied Electrochemistry[END_REF]. It is not only a question of the accessibility of the active site to the analyte but also possible unfolding phenomena when attached to a solid surface. For instance, Hampitak et al, studied the interaction and conformation of bovine serum albumin (BSA) adsorbed on graphene oxide and reduced graphene oxide. He observed denaturation on hydrophobic reduced graphene oxide by flattening the globular protein during the adsorption process while BSA remained intact when adsorbed on graphene oxide [START_REF] Hampitak | Protein interactions and conformations on graphene-based materials mapped using a quartz-crystal microbalance with dissipation monitoring (QCM-D)[END_REF]. Changes of these behaviours can certainly be expected when using other proteins or differently prepared graphene oxides. Besides the different proposed optimized Hummers methods [START_REF] Hummers | Preparation of Graphitic Oxide[END_REF], the origin of the graphitic carbon plays a crucial role for the properties of the obtained graphene oxide [START_REF] Kim | Building with graphene oxide: effect of graphite nature and oxidation methods on the graphene assembly[END_REF]. The same risks of reproducibility issues are present for graphene preparation independent on a top-down (exfoliation of graphite) or bottom-up (CVD synthesis) approach. There are several synthesis methods which all produces different kinds of graphenes in terms of layer amount and sizes, and quantity or nature of eventually introduced defects which needs adjustments of processing for each graphene source [START_REF] Whitener | Graphene synthesis[END_REF]. Carbon nanotube synthesis became industrial processes where homogeneous materials with similar properties are available even from different producers. Single walled, thin multi walled, thick multiwalled, or double/triple walled CNTs and the synthesis process, however, should still be taken into account [START_REF] Grobert | Carbon nanotubes -becoming clean[END_REF]. Therefore, for each bioentity, an adjusted protocol has to be established for every carbon nanomaterial.

Nonetheless, carbon (nano)materials appear to be very suitable for the immobilization of bioentities. On the one hand, their hydrophobic character allows stable interaction with hydrophobic amino acid residues or nucleotides in aqueous media. On the other hand, there are many efficient functionalization methods available to graft any desired chemical functions on the carbon surface to establish an optimal anchoring of biomolecules. The different immobilization techniques for biomolecules on carbon electrodes can be divided into covalent, non-covalent, or via supramolecular interactions including coordination on metal organic compounds as summarized in Figure 2.

Hydrophobic interactions

Within the non-covalent functionalization methods, the hydrophobicity of carbon is a clear advantage for targeted interaction with biomolecules. An accumulation of hydrophobic amino acid sidechains in proteins can form domains which adsorb easily on carbon surfaces in aqueous media without the necessity of pre-treatments [START_REF] Hofstee | Hydrophobic Effects in Adsorptive Protein Immobilization[END_REF]. A very nice example is Laccase from Trametes versicolor which is intensively used for enzymatic degradation of phenolic lignin and more recently, as cathodic biocatalyst in enzymatic fuel cell applications [START_REF] Goff | Recent progress in oxygen-reducing laccase biocathodes for enzymatic biofuel cells[END_REF]. This Laccase reduces oxygen to water at a trinuclear T2/T3 copper centre while oxidizing phenolic compounds at the separate mononuclear Type 1 copper centre. To capture these phenolic compounds, this T1 centre is close to a hydrophobic domain with an amino acid sequence containing aromatic and hydrophobic residues like phenylalanine, histidine, or leucine [START_REF] Blanford | A stable electrode for high-potential, electrocatalytic O 2 reduction based on rational attachment of a blue copper oxidase to a graphite surface[END_REF].(Figure 3) This domain therefore provokes oriented immobilization of this enzyme on hydrophobic surfaces that allows close contact of the T1 centre to the carbon electrode enabling direct electron transfers. This phenomenon could even be improved by attaching small hydrophobic or aromatic molecules onto the carbon surfaces [START_REF] Lalaoui | Hosting Adamantane in the Substrate Pocket of Laccase: Direct Bioelectrocatalytic Reduction of O 2 on Functionalized Carbon Nanotubes[END_REF][START_REF] Lalaoui | Direct Electron Transfer between a Site-Specific Pyrene-Modified Laccase and Carbon Nanotube/Gold Nanoparticle Supramolecular Assemblies for Bioelectrocatalytic Dioxygen Reduction[END_REF].

Figure 3: Wiring of laccase via hydrophobic interactions. Direct electron transfers can already be achieved by simple adsorption of laccase on the hydrophobic surface of nanocarbons. This effect can be improved with small molecules simulating phenols, the natural substrate of laccase.

The hydrophobic character of carbon nanomaterials represents also a challenge for the formation of bio-assemblies which needs to be done in aqueous media. This could firstly be demonstrated while using "high quality" SWCNTs with little surface defects [START_REF] Baur | Three-dimensional carbon nanotube-polypyrrole-[NiFe] hydrogenase electrodes for the efficient electrocatalytic oxidation of H 2[END_REF]. A mixture of a hydrogenase and methylviologen pyrrole monomer was drop casted on a SWCNT modified electrode. The aim was to entrap the hydrogenase inside the electrogenerated polypyrrol matrix to obtain mediated electron transfer during bioelectrocatalytic H 2 oxidation. Under the same conditions, "low quality" MWCNTs, with many oxygenated surface defects showed clearly enhanced electrocatalytic currents (300µA cm -2 ) than the SWCNT bioelectrodes (200µA cm -2 ). Considering the enhanced conductivity of SWCNTs compared to MWCNTs, this result might appear surprising. The reason for improved electrocatalysis for "cheap" MWCNT bioelectrodes could be revealed with scanning electron microscopy (Figure 4). With the SWCNT deposits, the aqueous monomer-enzyme solution remained at the surfaces and formed a thick polymer layer after electropolymerization. The defective surface of MWCNTs allowed a better penetration of the aqueous solution in the deposits and a homogeneous polymer cover was formed around the tubes thus reducing the distances for electron transfers, leading to improved electrocatalysis. More recently, the challenge to used superhydrophobic carbon nanotube forests for the formation of bioassemblies was faced by Singh et al. [START_REF] Singh | Functionalization of Contacted Carbon Nanotube Forests by Dip Coating for High-Performance Biocathodes[END_REF] The forests were initially modified with the amphiphile pyrene-bis-anthraquinone in an organic solvent to improve the wettability. Pyrene-bis-anthraquinone has shown to be an excellent hydrophobic anchor for oriented immobilization of laccase [START_REF] Bourourou | Supramolecular immobilization of laccases on carbon nanotube electrodes functionalized with (methylpyrenylaminomethyl)anthraquinone for direct electron reduction of oxygen[END_REF]. Here, these molecules further allowed the enzyme coating of the forests in depth to form an efficient biocathode.

Pi-stacking

A further advantage of sp 2 nanocarbon materials is the possibility to employ π-stacking interactions with biological compounds. In this context, a multihemic nitrite reductase adsorbed spontaneously on carbon nanotube modified electrodes via its heme units which are iron containing porphyrins. Such oriented immobilization enabled direct electron transfers during catalytic nitrite reduction [START_REF] Silveira | Enhanced direct electron transfer of a multihemic nitrite reductase on single-walled carbon nanotube modified electrodes[END_REF]. This easily formed and performing biosensors lacked long term stability which could be improved after electrogeneration of a protective cover using an amphiphilic pyrrole derivative mixed with clay. Similar approaches to modify CNT or graphene electrodes with heme containing enzymes like horseradish peroxidase (HRP) or cytochrome c were reported for the fabrication of electrochemical H 2 O 2 or nitrite biosensors [START_REF] Haldorai | Direct electrochemistry of cytochrome c immobilized on titanium nitride/multi-walled carbon nanotube composite for amperometric nitrite biosensor[END_REF][START_REF] Wang | A amperometric biosensor for hydrogen peroxide by adsorption of horseradish peroxidase onto single-walled carbon nanotubes[END_REF]. HRP became evenly of interest as cathodic biofuel cell catalyst in combination with H 2 O 2 producing enzymes like glucose oxidase [START_REF] Elouarzaki | Freestanding HRP-GOx redox buckypaper as oxygen-reducing biocathode for biofuel cell applications[END_REF][START_REF] Agnès | A double-walled carbon nanotube-based glucose/H 2 O 2 biofuel cell operating under physiological conditions[END_REF]. This bienzymatic setup is particular interesting for in vivo applications since these enzymes are less affected by interferants or inhibitors present in blood or extracellular liquids compared to the currently used multi copper enzymes.

The ability of porphyrins to π-stack on CNTs was firstly studied by Roquelet et al. who used a micelle-swelling technique to form assemblies between water insoluble porphyrins and CNTs in aqueous media for light harvesting [75] . Porphyrin functionalized CNTs can also act for oriented immobilization of the enzyme bilirubin oxidase (BOx). This enzyme is also a member of the multi copper enzyme family reducing oxygen to water at the T2/T3 centre by oxidizing bilirubin at the T1 centre, its natural substrate. Bilirubin is a linear tetra-pyrrole with two carboxylic acid functions at the inner pyrroles and is the metabolic breakdown product of heme. The Atanassov team firstly reported the oriented immobilization of BOx via functionalization of CNTs with bilirubin and or synthetic analogues leading to a drastic enhancement of electrocatalytic currents via direct electron transfers [START_REF] Lopez | Improved Interfacial Electron Transfer in Modified Bilirubin Oxidase Biocathodes[END_REF]. The cyclic form of bilirubin, protoporphyrin IX, the iron free form of heme, showed improved πstacking interactions related to the enhanced conjugated π system compared to its linear form. Even greater catalytic current densities could be obtained with this cyclic form of bilirubin [START_REF] Lalaoui | Fully Oriented Bilirubin Oxidase on Porphyrin-Functionalized Carbon Nanotube Electrodes for Electrocatalytic Oxygen Reduction[END_REF] and heme. This study also revealed the importance of carboxylic acid functions on the porphyrins. By having a detailed view on the protein structure around the T1 centre of BOx, an accumulation of basic amino acids (mainly histidine and arginine) can be observed [START_REF] Cracknell | Bilirubin oxidase from Myrothecium verrucaria: X-ray determination of the complete crystal structure and a rational surface modification for enhanced electrocatalytic O 2 reduction[END_REF]. Lalaoui et al. investigated the possibility of oriented immobilization of BOx only in presence of carboxylic acids [START_REF] Lalaoui | Diazonium Functionalisation of Carbon Nanotubes for Specific Orientation of Multicopper Oxidases: Controlling Electron Entry Points and Oxygen Diffusion to the Enzyme[END_REF]. CNTs were covered via diazonium grafting with carboxy naphthalene which led to similar electrocatalytic performances as with protoporphyrin IX showing that such prophyrins are not essential for oriented immobilization of BOx. Interestingly, in the same manner, CNTs were functionalized with amino naphthalene and a different redox system at lower potential was observed after immobilization of BOx. Considering a higher density of aspartic and glutamic acid residues close to the T2/T3 centre, it could be concluded that the enzyme was oriented with this catalytic centre to the CNT surface (Figure 5). Detailed studies about the electrocatalytic behaviour and electron transfer kinetics of these two catalytic sites were realized with site specific modified laccases with one pyrene function either close to the T1 centre or close to the T2/T3 centre [START_REF] Gentil | Efficiency of Site-Specific Clicked Laccase-Carbon Nanotubes Biocathodes towards O 2 Reduction[END_REF].

Covalent binding of biomolecules

Pyrene derivatives became the entities of choice for non-covalent (bio) functionalization of carbon nanotubes and graphene via π-stacking interactions [START_REF] Cao | Electrochemical and Theoretical Study of π-π Stacking Interactions between Graphitic Surfaces and Pyrene Derivatives[END_REF][START_REF] Bailey | A study of planar anchor groups for graphene-based single-molecule electronics[END_REF][START_REF] Zhang | Direct Measurements of the Interaction between Pyrene and Graphite in Aqueous Media by Single Molecule Force Spectroscopy: Understanding the π-π Interactions[END_REF]. Chen et al. firstly grafted peptidic biomolecules onto SWCNTs using a standard amide coupling strategy with pyrene butyric acid Nhydroxy succinimidyl ester (pyrene-NHS) [START_REF] Chen | Noncovalent Sidewall Functionalization of Single-Walled Carbon Nanotubes for Protein Immobilization[END_REF] modified CNTs. This covalent binding of biolmolecules on non-covalently functionalized nanocarbon materials is easy to realise at room temperature via simple drop casting or incubations without the need of sophisticated synthesis equipment. This made this functionalization method the privileged strategy for the development of graphene or CNT based biosensors [START_REF] Gupta | Recent advances in carbon nanotube based electrochemical biosensors[END_REF][START_REF] Mishyn | The holy grail of pyrene-based surface ligands on the sensitivity of graphene-based field effect transistors[END_REF]. This strategy also commonly used with carboxylic acid functionalized carbon materials. Such acid function can be introduced by acidic oxidation of the carbon materials [START_REF] Liang | Multi-walled carbon nanotubes functionalized with a ultrahigh fraction of carboxyl and hydroxyl groups by ultrasound-assisted oxidation[END_REF]. Another efficient method assuring the acid functionalization over the whole carbon nanomaterial surfaces is their attachment via diazonium coupling with 4-carboxyphenyl diazomium salts [START_REF] Pilan | Tailoring the performance of electrochemical biosensors based on carbon nanomaterials via aryldiazonium electrografting[END_REF].The common strategy is to activate these carboxylic acid functions with carbodiimides (dicyclohexyl carbodiimide, DCC, in organic solvents and 1-ethyl-3-(3dimethyllaminopropyl) carbodiimide hydrochloride, EDC in aqueous media). In aqueous solutions, terminal amines of proteins or amine modified DCA strands can directly be attached to the carbon surface while when working in organic solvent, the NHS ester must be formed before transferring to the aqueous system. Another way for covalent grafting biomolecules is the use of the crosslinking capacity of glutar aldehyde (1,5-Pentanedial). Aldehyde can spontaneously form imines with the amines of enzymes and other proteins and link them to an amine modified surface [START_REF] Melo | New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH[END_REF]. Amine functions can efficiently be attached by coupling diamines with the carboxylic acid functions of oxidized carbon materials, by coating with aminated polymers, or by pyrene amines [START_REF] Anzar | Carbon nanotube -A review on Synthesis, Properties and plethora of applications in the field of biomedical science[END_REF]. As a complementary covalent functionalization strategy to these common methods is the 1,3-Dipolar cycloaddition of azomethine ylides which allows a controlled grafting of multiple function for biomolecule immobilization and/or activation [START_REF] Dubey | Functionalized carbon nanotubes: synthesis, properties and applications in water purification, drug delivery, and material and biomedical sciences[END_REF][START_REF] Battigelli | Endowing carbon nanotubes with biological and biomedical properties by chemical modifications[END_REF].

Non-covalent binding of biomolecules (Host-guest interactions, coordination)

The immobilization of the bioreceptor unit by covalent grafting, however, is little controlled and can affect its biological activity and thus lowering the performances. In this context, targeted labelling of bioentities with anchor functions for immobilization or transduction is a powerful tool for improved biosensor performances. The most famous example is the avidin-biotin strategy which has some notable advantages compared to NHS coupling. The extremely specific and high-affinity interactions between four biotins and the glycoprotein avidin (association constant K a =10 15 L mol -1 ) [START_REF] Weber | Structural origins of highaffinity biotin binding to streptavidin[END_REF] allows to strong associations similar to covalent bonds. The principle is based on the modification of both, the transducer surface and the bioreceptor with biotin where each component can be modified, characterized, and stored until use under the respective optimal conditions. Related to this, many different bioreceptors modified with a biotin function are now commercially available. The biosensor can then be constructed via formation of an avidin layer on the transducer followed by the immobilization of the receptor unit by simple incubation in the respective solutions. The intermittent avidin layer further serve for passivation of the surface preventing non-specific binding of interfering biomolecules. This passivation is quite beneficial for immune or DNA sensors. For electrochemical enzyme biosensing, this intermittent protein layer has more a detrimental effect since the enzymatic transformed redox probes has hindered diffusion to the electrode surface.

An alternative to this is to immobilize biotinylated bioreceptors on β-cyclodextrin [START_REF] Holzinger | Biotin --cyclodextrin: a new host-guest system for the immobilization of biomolecules[END_REF] or nitrilotriacetic acid (NTA) [START_REF] Baur | Immobilization of biotinylated biomolecules onto electropolymerized poly(pyrrole-nitrilotriacetic acid)-Cu 2+ film[END_REF] modified surfaces (Figure 6). NTA-copper or nickel complexes are generally used to immobilize histidine tagged molecules and found firstly applications for the purification of biomolecules. The chelating effect of NTA forms strong Ni(II) or Cu(II) complexes which provide two available coordination sites which are occupied by water molecules [START_REF] Knecht | Oligohis-tags: mechanisms of binding to Ni2+-NTA surfaces[END_REF]. The imidazole function of histidine coordinates via its sp² nitrogen where this binding is strong enough for the immobilization of biological macromolecules but in presence of free imidazole, the competing interactions release the biomolecules and thus regenerate the surface. Biotin coordinates by its sulfur on copper or nickel NTA with approximately the same stability and this NTA/Cu 2+ /biotin system became a reliable immobilization strategy biosensing applications [START_REF] Baur | Immobilization of biotinylated biomolecules onto electropolymerized poly(pyrrole-nitrilotriacetic acid)-Cu 2+ film[END_REF][START_REF] Palomar | Controlled carbon nanotube layers for impedimetric immunosensors: High performance label free detection and quantification of anti-cholera toxin antibody[END_REF][START_REF] Singh | Non-covalently functionalized monolayer graphene for sensitivity enhancement of surface plasmon resonance immunosensors[END_REF]. Biotin and β-cyclodextrin form supramolecular inclusion complexes with an association constant of around 10² L mol -1 . This interaction is by far weaker than the biotin-avidin binding but strong enough to immobilize bioentities. Stronger and more common is the host-guest system β-cyclodextrinadamantan [START_REF] Buzzetti | Organic -cyclodextrin Nanoparticle: An Efficient Building Block Between Functionalized Poly(pyrrole) Electrodes and Enzymes[END_REF][START_REF] Haddad | Pyreneadamantane-β-cyclodextrin: an efficient host-guest system for the biofunctionnalization of SWCNT electrodes[END_REF] with an association constant of K a = 10 4 L mol -1 [START_REF] Granadero | Host-Guest Complexation Studied by Fluorescence Correlation Spectroscopy: Adamantane-Cyclodextrin Inclusion[END_REF]. However, β-cyclodextrin form inclusion complexes with a wide series of small molecules with sufficient hydrophobicity and appropriate sizes and even can act as surfactant for reduced graphene oxide [START_REF] Fritea | Design of a reduced-graphene-oxide composite electrode from an electropolymerizable graphene aqueous dispersion using a cyclodextrin-pyrrole monomer. Application to dopamine biosensing[END_REF]. Such non-specific behavior represents a disadvantage since proteins [START_REF] Lagiewka | Cyclodextrins-Peptides/Proteins Conjugates: Synthesis, Properties and Applications[END_REF] and DNA[104] in complex fluids can interfere and form competitive inclusion complexes removing the initially immobilized bioreceptor unit.

Further immobilization methods were proposed combining affinity and host-guest interactions with coordination chemistry by immobilizing pyrene modified bipyridyl ligands on carbon nanotube electrodes. β-cyclodextrin modified enzymes could be immobilized via inclusion of pyrenes of a trisbipyridyl iron complex [START_REF] Goff | Tris(bispyrenebipyridine)iron(II): a supramolecular bridge for the biofunctionalization of carbon nanotubes via -stacking and pyrene/-cyclodextrin host-guest interactions[END_REF]. A mixed ligand iron complex could be formed on CNTs with pyrene and biotin containing bipyridyl ligands [START_REF] Haddad | In situ synthesis of stable mixed ligand Fe 2+ complexes on bipyridinyl functionalized electrodes and nanotube supports[END_REF]. Biotinylated glucose oxidase could then be immobilized via the standard avidin bridges.

Once the protein or nucleotide based bioreceptor is chosen and the most appropriated immobilization strategy for nanostructured has to be determined. The desired anchor functions can be attached to nanocarbon surfaces via various methods. As already mentioned, pyrene is the mostly used function for non-covalent functionalization. A large variety of derivative is available which allows its modification with any chemical function capable to immobilize bioentities. Pyrene has further beneficial properties for nanocarbon based bioelectrochemical applications which were reviewed recently [START_REF] Holzinger | The versatility of pyrene and its derivatives on sp 2 carbon nanomaterials for bioelectrochemical applications[END_REF].

Within the noncovalent functionalization strategy, electrosynthesis of conductive polymers became an attractive method since functions for biomolecule immobilization can be introduced during synthesis as it is for pyrene derivatives [START_REF] Kaur | Review on nanomaterials/conducting polymer based nanocomposites for the development of biosensors and electrochemical sensors[END_REF]. Within these polymers, polypyrrole is of particular interest since its monomers can be electropolymerized in aqueous media. This allows in situ entrapment of enzymes in the polymer matrix as already discussed with the example of hydrogenase immobilization on different CNT supports [START_REF] Baur | Three-dimensional carbon nanotube-polypyrrole-[NiFe] hydrogenase electrodes for the efficient electrocatalytic oxidation of H 2[END_REF]. Beside this, this functionalization technique is quite efficient for homogeneous coating of porous nanostructures of CNTs and graphenes. Film thicknesses can easily be controlled by optimized charge transfer rates using chronoamperometry or chronopotentiometry. In the first case, the faradaic current is measured at a given potential until the desired charge is obtained. In the second case, the evolution of the potential is measured at given discharge current until the electropolymerization is completed. For optimal coverage of such polymer films on carbon nanostructures, organic solvents like acetonitrile or dimethyl formamide (DMF) allow better penetration and diffusion of the monomers to electropolymerize throughout the carbon electrode than aqueous media. The generated polymer film further allows improved wettability of the carbon nanostructures and represent an efficient platform for the immobilization of biomolecules.

Bioelectrochemical applications: Biosensors

Electrochemical transduction is an efficient tool for biosensing. Several techniques are available allowing a great choice of analytical methods [START_REF] Scholz | Electroanalytical Methods, Guide to Experiments and Applications[END_REF] (Figure 7). For instance, amperometry is quite appropriate for enzymatic biosensing since redox enzymes transform or produce electroactive redox species which can directly be detected at the bioelectrode [START_REF] Bollella | Enzyme based amperometric biosensors[END_REF]. By applying the corresponding redox potential, the current intensity can be directly correlated to the concentration of the analyte. The evaluation of the performances of amperometric biosensors is represented in a so-called calibration curve which is a function of the recorded current towards the analyte concentration. Such curves are divided in a linear part and a maximum current part. At high analyte concentrations, the enzymatic is not capable to transform the excess of these species and indicates the amount of immobilized biocatalysts on the electrode when its activity is known. Here, carbon nanomaterials have shown to provide an efficient extension of the electrode material related to excellent conductivity and the formation of porous structures resulting in a clear enhancement of the specific surface and thus the enhancement of immobilized biorecptors. The linear part represents all sensing kinetics concerning diffusion of the analyte or transduction of the recognition event (conversion of the generated redox probe at the electrode). This part is of interest for final sensor devices where the current intensity can directly be correlated to the analyte concentration. The higher the slope, the higher is the sensitivity of the sensors. For instance, the most applied example is enzymatic glucose sensing. Here, an enzyme like glucose oxidase, oxidised glucose into gluconic acid by reducing oxygen into hydrogen peroxide. This hydrogen peroxide is redox active and can be re-oxidized to oxygen and water on platinum electrodes at around 0.7 V vs AgAg/Cl [START_REF] Juska | A Critical Review of Electrochemical Glucose Sensing: Evolution of Biosensor Platforms Based on Advanced Nanosystems[END_REF]. Platinum with its catalytic properties is unfortunately needed since it can separate and distinguish the electrochemical oxidation of H 2 O 2 from water. In commercial point of care applications, platinum is generally avoided related to production costs where electron shuttles like ferrocene allows the transfer of the recovered electrons during oxidation from the FAD centre directly to the electrode and omit the need to detect the produced H 2 O 2 . In all cases, the measured currents are directly correlated to the glucose concentration in the linear part. There are further original and efficient transduction principles for glucose sensing which were nicely summarized by Pullano et al. [START_REF] Pullano | Glucose biosensors in clinical practice: principles, limits and perspectives of currently used devices[END_REF]. Highly sensitive enzymatic amperometric biosensors can be produced for the detection of phenolic pollutants like phenol, cresols, or dimethylphenols. The enzyme polyphenol oxidase oxidises the compounds forming ortho and para quinones which can electrochemically be detected at reducing potentials around -0.2 V vs Ag/AgCl. The formed hydroquinones are re-oxidized by the enzyme resulting in an amplification of the signal and allows low detection limits [START_REF] Raymundo-Pereira | Polyphenol oxidase-based electrochemical biosensors: A review[END_REF].

More sophisticated setups are needed when amperometric transduction is chosen for the detection of immune systems or DNAs. Since the recognition event between antibody-antigen or corresponding dana strands does not produce an electrochemical signal, a specific labelled secondary system (antigen, DNA strand) must be applied for signal capture. This more complex approach has the advantage of double recognition of two specific bioentities which reduces drastically interference and background effects and leads to more reliable signal capture. This principle of immunosensing is still the standard in clinical enzyme-linked immunosorbent assay (ELISA) tests [START_REF] Noji | Enzyme-based digital bioassay technology -key strategies and future perspectives[END_REF]. The formation of carbon nanotube or graphene deposits on electrodes clearly enhance the specific surface area but the narrow pore sizes prevent the efficiency of such double recognition events in depth of the deposit [115]. Singh et al. faced this issue by forming layer by layer assemblies with CNTs and magnetic nanoparticles [START_REF] Singh | Layer-by-layer scaffold formation using magnetic attraction between HiPCO® single-walled carbon nanotubes and magnetic nanoparticles: application for high performance immunosensors[END_REF]. The particles served here as spacers for improved diffusion of the receptor antigen of cholera, the analyte, anti-cholera toxin, and the glucose oxidase labelled secondary antibody throughout the structure. Different particle sizes were used to optimize the performances. It was shown that the highest sensitivity of 88 µA µg -1 mL cm -2 was obtained with 500 nm sized particles which is still one of the best performing immunosensor with amperometric transduction.

Cyclic voltammetry (CV) is a powerful technique to modify and to characterize the resulting electrodes. For instance, by cycling within an appropriate potential range, functional monomers can be electropolymerized forming thin films with chemical functions for the immobilization or entrapment of the bioreceptors [START_REF] Cosnier | Encyclopedia of Applied Electrochemistry[END_REF]. Such modified electrodes can then be characterized about diffusion and electron transfer kinetics through the formed films. For biosensing application, cyclic voltammetry is of interest when the product of an enzymatically formed product changes locally the pH value and thus the potential of the electrochemical probe. For immune or DNA sensing, the diffusion of an electrochemical probe is reduced after the recognition event since the receptor-analyte layer hinders the access to the electrode surface. One disadvantage of cyclic voltammetry is that not only redox events are recorded but also capacitive charging and discharging which results in low resolution of the current intensities. Carbon nanomaterials are high capacitive and to avoid a strong capacitive hysteresis, very slow scan rates have to be chosen. An alternative solution is the technique of differential pulse voltammetry (DPV). Here, short voltage pulses are applied where two current intensities are recorded before and at the end of the pulse. The subtracted current intensities for the whole potential range allow to measure only the faradaic current without the disturbing capacity. Electrochemical impedance spectroscopy (EIS) is more appropriate for immune-and DNA sensing since the signal of the recognition event can be recorded without such supplemental labelling steps [START_REF] Randviir | A review of electrochemical impedance spectroscopy for bioanalytical sensors[END_REF]. EIS records capacitance and interfacial electron transfer resistance during the recognition event using an alternative current with small amplitudes around a redox probe potential at a wide range of frequencies. When the immobilized antibody or DNA stand recognizes its counterpart, and electrochemical probe is retained for its diffusion to the electrode surface by the formation of an increased layer thickness or conformational changes causing higher electron transfer resistances. This technique is extremely sensitive and allows detection limits down to the femtomolar range [START_REF] Baur | Label-free femtomolar detection of target DNA by impedimetric DNA sensor based on poly(pyrrole-nitrilotriacetic acid) film[END_REF]. There are little examples reporting the use of nanostructured carbonmaterials for impedimetric biosensing. Most examples confine these materials in polymer matrices [START_REF] Terti | Electrochemical Impedance Studies on Single and Multi-Walled Carbon Nanotubes&#8212[END_REF][START_REF] Dong | A label-free electrochemical impedance immunosensor based on AuNPs/PAMAM-MWCNT-Chi nanocomposite modified glassy carbon electrode for detection of Salmonella typhimurium in milk[END_REF][START_REF] Zhou | Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films[END_REF][START_REF] So | Electrical Impedance Properties of Carbon Nanotube Composite Electrodes for Chemical and Biosensor[END_REF] which improves the interfacial electron transfer but omits to profit from the enhanced specific surface of this material.

The drawback is that deposits of CNTs or graphene form randomly nanostructures with slight differences of the final surface morphologies. The high sensitivity of EIS is strongly related to the surface morphology and even slight changes affect drastically the repeatability and reliability. To circumvent this effect, and to provide identic sensing elements, vertically aligned CNTs were used for impedimetric cancer cells [START_REF] Abdolahad | A vertically aligned carbon nanotube-based impedance sensing biosensor for rapid and high sensitive detection of cancer cells[END_REF]. EIS immunosensors were reported by Hafaiedh et al. who used air brush techniques to form homogeneous films of oxidized CNTs to form an impedimetric immune sensor for Rabbit IgG [124]. Palomar et al. formed thin films by filtrating highly diluted aqueous CNT dispersion over cellulose ester filter membranes. These films can then be transferred to electrode supports by dissolution of the filter in acetone. The resulting EIS biosensor for anti-cholera toxin detection showed great reproducibility and provides a linear range over more than eight orders of magnitude [START_REF] Palomar | Controlled carbon nanotube layers for impedimetric immunosensors: High performance label free detection and quantification of anti-cholera toxin antibody[END_REF].

Bioelectrochemical applications: Enzymatic Biofuel cells

Besides electrochemical enzyme biosensing, the possibility to use biocatalytic enzymes to oxidize a "fuel" like glucose or hydrogen, and to reduce an oxidizer like oxygen or hydrogen peroxide, to assemble these two "biosensors" to produce electric power, became a fascinating research field. Such biofuel cells can produce electric energy using non-toxic and non-polluting fuels and the biological energy production can take place at neutral pH, at room temperature, and in complex media due to the high specificity of enzymes. [125] . This technology gained high interest when a glucose biofuel cell design could harvest few µW of power from a grape [126]. Since glucose and oxygen are both present in body fluids, the idea of powering implantable devices like pace makers or insulin pumps by such biological fuel cells but the strict requirements, especially operational life times, are still far to be met [START_REF] Cosnier | Beyond the hype surrounding biofuel cells: What's the future of enzymatic fuel cells?[END_REF]. However, the main critics for enzymatic biofuel cells are their limited power output and operational life time. Katz et al., one main actor of enzymatic biofuel cell research over the last decades, recently published a very critical review article about this topic where the authors cannot imagine practical applications with industrial interest [START_REF] Katz | Fuel Cells and Biofuel Cells: From Past to Perspectives[END_REF]. It is true that enzymatic fuel cell will never be capable to face the steady increasing energy need. However, this technology has realistic potential in the field of low power consuming devices for single or short time use before disposal like pregnancy, ovulation or other point of care tests [START_REF] Abreu | Towards ecofriendly power sources: In series connected glucose biofuel cells power a disposable ovulation test[END_REF]. Such disposable medical devices are often powered by coin or button cell batteries which is the most polluting component. Biofuel cells meet the power demand of these devices and can replace the coin cell batteries in the future for a series of devices.

The challenges in biofuel cell research are not only to immobilize the enzymes but also to establish an electron transfer with the electrode. Such electron transfers should be at low overpotentials since the power output is directly related to the current density and the voltage. In the optimal case, direct electron transfers can be established between the redox center of the enzyme and the electrode as mentioned with the examples of multicopper enzymes. Concerning direct electron transfers with flavin adenine dinucleotide (FAD) dependent enzymes like glucose oxidase, the Bartlett team clearly demonstrated that all reports about direct oxidoreduction of the FAD co-factor is in fact the redox signature of free, non-catalytic, FAD, commonly found in commercialized enzyme samples [START_REF] Bartlett | There is no evidence to support literature claims of direct electron transfer (DET) for native glucose oxidase (GOx) at carbon nanotubes or graphene[END_REF]. In fact, the catalytic active cofactor is deeply embedded inside the protein which is further covered with a carbon hydrate shell. The distance between the redox center and a conductive surface is therefore too high which makes direct electron transfers via tunneling more than improbable [START_REF] Page | Natural engineering principles of electron tunnelling in biological oxidation-reduction[END_REF].The only credible example of direct electron transfer with catalytic FAD of glucose oxidase from Aspergillus niger was shown by Courjean et al. [START_REF] Courjean | Deglycosylation of Glucose Oxidase for Direct and Efficient Glucose Electrooxidation on a Glassy Carbon Electrode[END_REF]. Here, the oligo saccharide layer could complete be removed thus reducing the distance between the active site and the electrode and thus enabling direct electron transfers with this modified enzyme. For native glucose oxidase and other FAD dependent enzymes, however, so-called mediated electron transfer is necessary. Such mediation is achieved by using small redox active molecules which can shuttle electrons between the enzyme's redox center and the electrode. The redox potential of such electron shuttle has to be chosen carefully since a compromise between high overpotentials with high currents but loss of the cell voltage and low overpotential with low currents, but higher cell voltage has to be accepted (Figure 8). However, Tsuruoka et al. studied the mediated electron transfer rates with different organic redox mediators [START_REF] Tsuruoka | Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors[END_REF]. He concluded that the correlation between electrocatalytic currents and the redox potential is not evident since other factors like functional groups, size, or charges of such mediators also influence the transfer kinetics. The "best" mediator has to be experimentally determined for each enzyme even when it has the same co-factor. Figure 8: illustrations of an enzymatic bioanode and a biocathode with optional mediated electron transfers leading producing electrocatalytic current densities at distinguish voltages and consequently power.

Carbon nanomaterials play a crucial role for the performance of biofuel cells. After a first proof of concept using enzymes for the production of energy in 1964 [START_REF] Yahiro | Bioelectrochemistry: I. Enzyme utilizing bio-fuel cell studies[END_REF], there were little activities in this research field until the late 1990's where great improvements were achieved in enzyme wiring [135]and the first use of CNTs [START_REF] Guiseppi-Elie | Direct electron transfer of glucose oxidase on carbon nanotubes[END_REF]. It is well accepted that the tubular structure of CNTs is beneficial to enhanced access to the redox center of the enzymes and this material is still mostly used in this research topic. However, the toxicity of CNTs as aerosols is compared with asbestos fibers. Even when up to date only one class of stiff and long MWCNTs has been classified as a possible human carcinogen when inhaled [START_REF] Fukushima | Carcinogenicity of multi-walled carbon nanotubes: challenging issue on hazard assessment[END_REF][START_REF] Kane | The asbestos-carbon nanotube analogy: An update[END_REF] and a vast amount of different CNTs is still not classified yet, safety and security is of predominant importance for existing and future CNT based products where the release of CNTs as aerosols even after disposal must totally be excluded. Furthermore, compared to other carbon nanomaterials, the production costs are still relatively high despite wellestablished mass production methods. In this context, clear progresses were achieved using other nanostructured carbons like Ketjen/carbon black or graphene [139][START_REF] Minteer | New materials for biological fuel cells[END_REF]. Carbon black in general is composed of amorphous carbon micro/nanoparticles with conductive and insulating domains. Within this category, Ketjen and Vulcan black provides improved conductivities and nanosized structures and are therefore prominent alternatives for CNTs foe biofuel cell applications [START_REF] Bandodkar | Wearable Biofuel Cells: A Review[END_REF]. Targeted synthesis of carbon particles with controlled nanopore sizes were proposed for biofuel cell applications by the Tsujimura team [START_REF] Inagaki | Templated mesoporous carbons: Synthesis and applications[END_REF]. These particles are formed by annealing of a carbon precursor, mostly polyvinyl alcohol, in presence of magnesium oxide particles with controlled size and shape. After the carbonization process these MgO particles can easily be removed by dissolution in diluted acids leaving identic pores in the particles which has been shown to improve the operational lifetime of enzymes combined with efficient electron transfer kinetics [START_REF] Tsujimura | Exceptionally High Glucose Current on a Hierarchically Structured Porous Carbon Electrode with "Wired" Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase[END_REF]. Beside the tremendous impact of graphene materials in bioelectrochemical applications, this carbon material shows less efficiency for biofuel cell technologies. Within the different graphene materials, reduced graphene oxide appears to be most promising, but the performances are generally far below compared to the other carbon nanomaterials as discussed above [START_REF] Tang | Development of graphene-based enzymatic biofuel cells: A minireview[END_REF].

Conclusion

Considering the vast number of available carbon nanomaterials, immobilization techniques, and electrochemical transduction methods, the optimal biosensor setup might be hard to determine for an analyte of interest. However, there are some standard advices which facilitates the choice. For the electrochemical detection of small molecules, enzymatic biosensors or aptasensors are mostly used. Several immobilization techniques were discussed to for a reliable enzyme-based sensing element via simple adoption, forming electrostatic, or hydrophobic interactions. The enzymatic reactions can form electroactive species which can be transduced via amperometry of cyclic voltammetry. When no electroactive species is formed, electron transfers between the enzyme and the electrode can be achieved by direct electron transfers or by using redox mediators. Cyclic voltammetry gives most information about the electrocatalysis but related to the high capacitance of carbon materials, slow scan rates must be used. For the detection of diseases, immune systems or DNA are the sensing element of choice. Here, more sophisticated immobilization is recommended to avoid inhibition or loss of biological activities. Electrochemical impedance spectroscopy is here a powerful transduction technique since it allows label free detection. Carbon nanomaterials by nature provide an extended electroactive surface area for enhanced immobilization of generally bulky bioreceptors. The choice of which nanomaterial is most appropriate for which receptor unit or electrochemical transduction remains, however, controversial.

Fig. 1 :

 1 Fig. 1: Most studied carbon allotropes carbon allotropes. A) helical CNTs, B) fullerenes, C) diamond, D) fishbone-type carbon nanofibers, E) nanohorns, F) graphene, G) MWCNTs, H) SWCNTs (in bundle), I) graphene quantum dots, and K) carbon quantum dots.

Figure 2 :

 2 Figure 2 : Currently used functionalization methods for biomolecule immobilization. A) non-covalent functionalization with pyrenes, B) covalent functionalization via diazonium coupling, C) coating with polymers (here, electrogenerated poly pyrrolefilms), and D) oxidation of the carbon materials with subsequent activation of carboxylic acid defects via activated esters. These methods allow the attachment of various biomolecule linker functions like a) NTA, b) activated succinimidyl ester, c) alkynes for "click" coupling, d) biotin for the cross linking with avidin bridges, e) ionic interaction with defectcarboxylates

Figure 4 :

 4 Figure 4: Electrochemical coating with viologen polypyrrole in presence of a hydrogenase on A) SWCNTs and B) MWCNTs.

Figure 5 :

 5 Figure 5: Controlled orientation of Box via targeted amine or carboxyl functionalization of CNTs wiring either the T1 centre or the T2/T3 centre.

Figure 6 :

 6 Figure 6: Immobilization of bioreceptor units by coordination of A) biotin or B) histitine tagged bioentities on NTA-Cu 2+ modified nanocarbon based electrodes. C) Historical immobilization strategy using the biotin-avidin-biotin system.

Figure 7 :

 7 Figure 7: Common transduction techniques for electrochemical biosensing: A) example of the operational mode of enzymatic biosensors where the analyte is oxidized or reduced producing a redox active species which can be in some cases the analyte itself. Or, the involved electrons can be transferred to the electrode. The redox processes at the electrodes can then be transduced via e.g. B) Cyclic voltammetry, C) Aperometry, and D) Differential pulse voltammetry. E) The recorded current evolution allows to establish a calibration curve.