FAD-dependent Glucose Dehydrogenase Research Articles

Improvement of substrate specificity of the direct electron transfer type FAD-dependent glucose dehydrogenase catalytic subunit

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing—including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme’s high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its activity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH’s small subunit and catalytic subunit (BcGDHγα), in complex with D-glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted, site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475. The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in Vmax towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5 mM) of 4.19 U mg−1, as compared to the wild type, 86.6 U mg−1. This resulting increase in specificity lead to an 81.7 gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9 gal/glc % activity at 5 mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose.

Designing a high-potential metal-free viologen-based redox polymer for effective wiring of FAD-dependent glucose dehydrogenase

Metal-free organic redox mediators, whose redox potentials can be tuned according to the intended enzyme system and application, are emerging as alternative mediators for the design of improved enzyme-based biosensors and biofuel cells. We report a novel class of metal-free redox polymer containing a polymer-tethered dicyano viologen mediator for the immobilization of FAD-dependent glucose dehydrogenase (FAD-GDH) on graphite electrodes. Incorporation of strong electron-withdrawing cyano groups to the viologen moiety ensures the formation of high potential viologen-based redox polymer with an envisaged pH-independent redox potential close to 50 mV (vs Ag /AgCl/ 3 M KCl) and a negligible impact on oxygen reduction. The bioelectrode comprising of newly designed redox polymer and FAD-GDH showed enhanced electrocatalytic activity in the presence of glucose, confirming effective wiring of FAD-GDH, if embedded within the redox polymer.

Facilitated Enzymatic Chain Reaction-Based Bioelectrocatalysis via Solid Binding Peptide-Guided Bienzyme Co-Immobilization Strategy

The multienzyme complex in biological systems are highly ordered so that intermediate molecules occurred during enzymatic sequential reaction are efficiently delivered to downstream enzymes without diffused to bulk phase. Recently, the cascadic multienzymes have been adopted to enzymatic electrocatalytic platform to be applied for enzyme-based bioelectronics such as enzyme fuel cell, biosensors, and electrosynthetic system. This cascadic enzyme-electrode, in which interfacial electron transfer (ET) occurs concurrently with inter-enzyme chain reaction, has been regarded promising for the advancement of enzyme-based bioelectronics performance. However, co-regulation of interfacial electrical connection and inter-enzyme chain reaction efficiency has been known to be highly challenging due to systematic complexity. In this context, the generalized enzyme immobilization tool is significantly needed to be developed to control inter-enzyme and enzyme-electrode interface concurrently.Herein, enzyme cascade-based direct bioelectrocatalytic system has been constructed by immobilizing enzymes using the solid binding peptide (SBP) linker that can control surface-orientation of enzymes on electrode. Here, invertase (INV) and FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) were utilized as upstream- and downstream enzyme so that the sucrose hydrolysis (at INV) and glucose oxidation (at GDHγα) is concomitantly occurred. Especially, the GDHγα that is direct electron transfer (DET)-capable oxidoreductase, has bi-function that are downstream catalysis and transport of produced electrons toward electrode that cause bioelectrocatalytic current signal. To immobilize enzymes and control relative orientation of coupling enzymes, the SBP linker was tethered various termini (C-, N-, or both termini) of INV when SBP fusion site of GDHγα was fixed to C-terminus of GDH α subunit to enable efficient interfacial DET, based on previous study. Therefore, the inter-enzyme relative orientation dependent chain reaction efficiency was evaluated with resulting DET-based electrocatalytic current. In the result, it was found that the interfacial DET at GDHγα-electrode could be affected by binding conformation of co-immobilized enzyme, fusion INV. Most importantly, the chain reaction efficiency between INV and GDHγα was revealed to be diverse depending on different relative orientation determined by SBP tethering sites in enzymes. The intermediate delivery route was changed by relative positioning of coupling active sites, affecting overall cascade reaction rate. Taking into account the factors related with interfacial DET and intermediate delivery, precise design of bienzymatic electrode is indeed necessary in order to introduce SBP-tethering technique to cascadic enzyme-derived direct electrocatalytic platform. Figure 1

Electrochemical and biosensing properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens

We investigated the bioelectrochemical properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens (TvGDH) and its electrochemical behaviour when immobilized on a graphite electrode. TvGDH was recently shown to have an unusual substrate spectrum and to prefer maltose over glucose as substrate, and hence could be of interest as recognition element in a maltose sensor. In this study, we determined the redox potential of TvGDH, which is –0.268 ± 0.007 V vs. SHE, and advantageously low to be used with many redox mediators or redox polymers. The enzyme was entrapped in, and wired by an osmium redox polymer (poly(1-vinylimidazole-co-allylamine)-{[Os(2,2′-bipyridine)2Cl]Cl}) with formal redox potential of +0.275 V vs. Ag|AgCl via poly(ethylene glycol) diglycidyl ether crosslinking onto a graphite electrode. When the TvGDH-based biosensor was tested with maltose it showed a sensitivity of 1.7 μA mM−1cm−2, a linear range of 0.5–15 mM, and a detection limit of 0.45 mM. Furthermore, it gave the lowest apparent Michaelis-Menten constant (KM app) of 19.2 ± 1.5 mM towards maltose when compared to other sugars. The biosensor is also able to detect other saccharides including glucose, maltotriose and galactose, these however also interfere with maltose sensing.

Cytochromes as electron shuttles from FAD-dependent glucose dehydrogenase to electrodes

Cytochromes are a large and well-known group of electron transfer proteins, but only very few are used in bioelectrochemistry, e.g. to serve as electron shuttles between biocatalysts and electrodes. There, cytochromes generate an artificial electron transfer pathway if their structural and electrochemical properties are fitting. Cytochromes considered suitable are small and feature an exposed heme cofactor, a suitable redox potential, an electrochemically reversible redox process, sufficient protein production, and a fast electron transfer between other enzymes and hemoproteins. Out of 850 screened hemoproteins, 58 cytochromes met the requirements to function as an electron shuttle. They can be classified into five distinguished protein folds. Four representative b-type and three c-type cytochromes were selected for further analyses. The proteins were obtained in a yield of 0.075 to 375 mg L-1 in Escherichia coli or Komagataella phaffii. The electrochemical reversibility on 1-thioglycerol functionalized gold electrodes of all cytochromes was confirmed. The pH-dependent redox potentials range from -50 mV to +298 mV vs. SHE at pH 7.0. The electronic wiring ability was tested using the biocatalyst FAD-dependent glucose dehydrogenase (GDH). Cytochrome reduction by the GDH was monitored by spectrophotometry and amperometry. c-Type cytochromes showed a faster electron uptake from GDH than b-type cytochromes. Complementary surface charges at the cytochrome/GDH interface were not a predictor for a fast electron transfer but a higher difference in the redox potential of the cofactors was. This study supports the design of bioelectrocatalysts with high electron transfer rates, which are of great interest in 3rd generation biosensors and for direct bioelectrocatalysis.

Redox potential of FAD-dependent glucose dehydrogenase

The redox potential is important to rationally employ oxidoreductases as bioelectrocatalysts to minimize electroactive interferences and maximize current density or cell voltage. For many FAD-dependent enzymes redox potentials are published, but not for FAD-dependent glucose dehydrogenase (GDH) from the glucose-methanol-choline (GMC)-oxidoreductase family, which are widely used in glucose biosensors. GDH is contacted via redox mediators or redox polymers and features a reasonably high substrate specificity and oxygen insensitivity. Here we report the redox potential of Glomerella cingulata GDH that was determined by two methods. Spectroelectrochemically we obtained a reduction potential of −0.265 ± 0.003 V vs SHE and with the xanthine oxidase assay using Janus green B a potential of −0.267 ± 0.016 V vs SHE. The determined redox potential of GcGDH differs greatly from that of Aspergillus niger glucose oxidase (−0.080 V vs SHE) despite an almost similar protein fold as GcGDH. Taking this excitingly low redox potential of GcGDH to develop optimized redox mediators and redox polymers for the fabrication of glucose oxidizing bioanodes with a low operating potential can drastically reduce the susceptibility of glucose biosensors towards electroactive substances or increase the cell voltage in biofuel cells.

Catalytic Biosensors Operating under Quasi-Equilibrium Conditions for Mitigating the Changes in Substrate Diffusion.

Despite the success of continuous glucose measuring systems operating through the skin for about 14 days, long-term implantable biosensors are facing challenges caused by the foreign-body reaction. We present a conceptually new strategy using catalytic enzyme-based biosensors based on a measuring sequence leading to minimum disturbance of the substrate equilibrium concentration by controlling the sensor between "on" and "off" state combined with short potentiometric data acquisition. It is required that the enzyme activity can be completely switched off and no parasitic side reactions allow substrate turnover. This is achieved by using an O2 -independent FAD-dependent glucose dehydrogenase embedded within a crosslinked redox polymer. A short measuring interval allows the glucose concentration equilibrium to be restored quickly which enables the biosensor to operate under quasi-equilibrium conditions.

Wiring of bilirubin oxidases with redox polymers on gas diffusion electrodes for increased stability of self-powered biofuel cells-based glucose sensing

A new redox polymer/bilirubin oxidase (BOD)-based gas diffusion electrode was designed to be implemented as the non-current and non-stability limiting biocathode in a glucose/O2 biofuel cell that acts as a self-powered glucose biosensor. For the proof-of-concept, a bioanode comprising the Os-complex modified redox polymer P(VI-co-AA)-[Os(bpy)2Cl]Cl and FAD-dependent glucose dehydrogenase to oxidize the analyte was used. In order to develop an optimal O2-reducing biocathode for the biofuel cell Mv-BOD as well as Bp-BOD and Mo-BOD have been tested in gas diffusion electrodes in direct electron transfer as well as in mediated electron transfer immobilized in the Os-complex modified redox polymer P(VI-co-AA)-[Os(diCl-bpy)2]Cl2. The resulting biofuel cell exhibits a glucose-dependent current and power output in the concentration region between 1 and 10 mM. To create a more realistic test environment, the performance and long-term stability of the biofuel cell-based self-powered glucose biosensor has been investigated in a flow-through cell design.

Conformational Change of His506 Residue in Reduce Enzyme of FAD-Dependent Glucose Dehydrogenase from A. oryzae

Conformational Change of His506 Residue in Reduce Enzyme of FAD-Dependent Glucose Dehydrogenase from <i>A. oryzae</i>

Peptide sequence-driven direct electron transfer properties and binding behaviors of gold-binding peptide-fused glucose dehydrogenase on electrode

SummaryOriented enzyme immobilization on electrodes is crucial for interfacial electrical coupling of direct electron transfer (DET)-based enzyme-electrode systems. As inorganic-binding peptides are introduced as molecular binders and enzyme-orienting agents, inorganic-binding peptide-fused enzymes should be designed and constructed to achieve efficient DET. In this study, it is aimed to compare the effects of various gold-binding peptides (GBPs) fused to enzymes on electrocatalytic activity, bioactivity, and material-binding behaviors. Here, GBPs with identical gold-binding properties but different amino acid sequences were fused to the FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) to generate four GDHγα variants. The structural, biochemical, mechanical, and bioelectrochemical properties of these GDHγα variants immobilized on electrode were determined by their fused GBPs. Our results confirmed that the GBP type is vital in the design, construction, and optimization of GBP-fused enzyme-modified electrodes for facile interfacial DET and practical DET-based enzyme-electrode systems.

Photo-Induced Signal Chains at Enzyme Electrodes

During the last decade photoelectrochemical devices which couple biochemical reactions to light-sensitive electrodes have gained increasing research interest. These kind of light-triggered systems have the inherent advantage that an additional tool - the light - can be used to control the reactions at a sensing electrode. This allows a light-directed read-out, but can also be used to change the energetic situation on the surface resulting in advantageous potential behavior [1].Enzymes as biocatalysts provide the advantage of a catalytic analyte conversion and specificity in this reaction. In order to couple them to light-sensitive electrodes different approaches have used.Besides the use of co-substrates or products [2], we have been focusing on the use of mediators for this interaction [3] or the establishment of a direct electron transfer of the enzyme to the light- sensitive entity [4]. Here we report on the use of two different types of polymers for establishing the electrical contact.In order to exploit the advantages of a high surface area we have constructed 3D electrodes which can be rather easily prepared by a template approach combined with spin coating as deposition method..Thus, the thickness of the electrode structure can be adjusted in a defined way [5,6].In a first approach 3D TiO2 of inverse opal character has been sensitized by a conducting polymer – sulfonated polyaniline. The polymer has here two functions: It ensures the light sensitivity in the visible range and also provides an interface at which the enzyme can exchange electrons. Advantageously the current signal can be gained at rather low potential. Furthermore, the response can be tuned by the dimensions of the 3D electrode demonstrating the scalability of the approach.As a further advancement we have used a redox polymer in order to couple FAD-dependent glucose dehydrogenase to the electrode. Here PbS QDs have been generated directly onto a 3D TiO2 electrode and characterized by TEM and photoelectrochemistry. The system allows the generation of a defined anodic photocurrent, which can be significantly enhanced by the biocatalytic conversion of glucose. Here the substrate dependent current signal starts at about -054V vs Ag/AgCl.Such electrode systems can find application in light-triggered sensing, but also for bioenergetics since the photocatalytic process can be “feeded” by electrons from the biocatalytic conversion.

Insights into carbon nanotube-assisted electro-oxidation of polycyclic aromatic hydrocarbons for mediated bioelectrocatalysis.

A series of polycyclic aromatics, naphthalene, phenanthrene, perylene, pyrene, 1-pyrenebutyric acid N-hydroxysuccinimide ester (pyrene NHS) and coronene, were immobilized via π stacking on carbon nanotube (CNT) electrodes and electro-oxidized in aqueous solutions. The obtained quinones were characterized and evaluated for the mediated electron transfer with FAD dependent glucose dehydrogenase during catalytic glucose oxidation.

Freestanding biopellet electrodes based on carbon nanotubes and protein compression for direct and mediated bioelectrocatalysis

The fabrication of bioelectrocatalytic protein electrodes by the simple compression of carbon nanotube and protein powders was investigated using a series of proteins including bilirubin oxidase (BOx), bovine serum albumin (BSA), catalase, cytochrome C (Cyt C), diaphorase, FAD-dependent glucose dehydrogenase (FAD-GDH), galactose oxidase (GAOx), glucose oxidase (GOx), horseradish peroxidase (HRP), laccase and urease. The isoelectric points (pI) of the proteins ranged from 3.5 to 12 and the molecular weights ranged from 12.4 to 480 kDa. The compression of diaphorase, laccase, BOx, FAD-GDH, catalase and urease gave mechanically stable biopellets in 0.1 M phosphate buffer pH 7.4. For Cyt C, BSA, GAOx, GOx, and HRP, contact with buffer destabilised the biopellet and induced a fast destruction of the composite. In parallel, stable redox pellets were obtained with differently charged redox mediators: hexaammineruthenium (III) chloride, potassium hexacyanoferrate or 1,4-naphthoquinone. Several parameters were explored to shed new light on the factors that determine biopellet stability. Stable biopellets capable of direct and mediated electron transfer were subsequently elaborated, for O2 reduction and glucose oxidation, respectively, based on the simplified procedure that does not require stabilising protection membranes or holders on the contrary to existing methods.

Electrochemical Aptasensor Employing Enzyme-Aptamer One-to-One Complex Showing Quasi-Direct Electron Transfer Ability

1. Introduction In this study, we report an innovative electrochemical aptasensor based on the labeling aptamer with quasi-direct electron transfer (quasi-DET)-type redox-enzyme, by developing a breakthrough technological platform to prepare one-to-one complex of enzyme and aptamer.The chronoamperometric-based electrochemical aptasensor is an ideal sensor principle using aptamers for biomolecular recognition considering signal amplification by the labelled redox-enzyme. However, conventional methods to label aptamers with enzymes are chemical modification using cross-linking reagents, which often result in uncontrollable multimer-complexes. Therefore, the method to prepare enzyme-aptamer one-to-one complexes which yields in reproducible and designed enzyme-aptamer complex is technologically challenging. We here report the development of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 2. Methods Enzyme-aptamer one-to-one complex was constructed utilizing monomeric streptavidin (mSA) and biotin complex formation [1] [2]. We used FAD-dependent glucose dehydrogenase (GDH) from Aspergillus flavus as an aptamer labelling enzyme, and anti-vascular endothelial growth factor (VEGF) aptamer, as to proof of this conceptional study. VEGF is a biomarker for cancer diagnosis. We created the recombinant fusion protein of GDH and mSA (GDH-mSA). The combination of GDH-mSA with biotinylated anti-VEGF aptamer results in their one-to-one complex. To evaluate the formation of the complex and detect VEGF using GDH activity, we performed enzyme-linked aptamer assay (ELAA). Next, GDH-mSA was evaluated as a label enzyme for electrochemical aptasensor, by chronoamperometry analysis using one-to-one complex of GDH-mSA and thiolated and biotinylated aptamer on the gold electrode. Before the analysis, we modified GDH-mSA covalently with phenazine ethosulfate (PES) which was a redox probe enabling redox-enzymes to possess quasi-DET ability [3]. The formation of the enzyme-aptamer one-to-one complex on the electrode and quasi-DET ability of PES-modified GDH-mSA was evaluated. 3. Results GDH-mSA showed dye-mediated GDH activity, indicating the fusion of mSA did not negatively affect the catalytic activity. As a result of ELAA, GDH-mSA showed remarkable GDH activity in the presence of VEGF and biotinylated anti-VEGF aptamer. This indicated one-to-one complex of GDH-mSA and biotinylated anti-VEGF aptamer was formed as we designed, and showed specific recognition and binding with VEGF. Chronoamperometry analysis was carried out, and the signal was increased strikingly upon the addition of glucose only with the gold electrode immobilizing PES-modified GDH-mSA with biotinylated aptamer. Unmodified GDH-mSA did not show quasi-DET. These results indicated our success in the construction of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 4. Conclusions In this study, to circumvent the formation of uncontrollable multimer-complexes of enzymes and aptamers, we created enzyme-aptamer one-to-one complex based on the mSA-biotin interaction. Thus designed enzyme-aptamer one-to-one complex was prepared without losing both characteristics and acknowledging both benefits. These results suggested that GDH-mSA and biotinylated aptamer complex can be applied for the development of quasi-DET-type aptasensor by simply modifying GDH-mSA with PES. These benefits of utilizing GDH-mSA combined with aptamer will lead to the further application of aptamer-based biosensing of various targets. 5.

The Continuous 3 Month Operation of Open Circuit Potential Based Glucose Sensor Employing Direct Electron Transfer Type Fad Dependent Glucose Dehydrogenase

Introduction To control glycemic levels of patients with type 1 diabetes, continuous monitoring of blood glucose concentrations is inevitable for the subsequent infusion of insulin. Since the amount of insulin being supplied is calculated based on the continuous glucose monitoring (CGM) results, CGM is considered as the core component for the continuous and accurate diabetes management. With the increase of the demands on convenient and user-friendly system, much accurate and stable sensing systems are required. Current commercially available CGM sensors employing amperometric enzyme sensor principles, are operated under continuous potential application to the glucose oxidoreductases and redox mediators, to keep their continuous electrochemical-catalytic reaction. Under such harsh conditions, inactivation of enzymes and mediators is unavoidable during continuous operation. Additionally, potential application to the system may cause oxidation of interfering substances directly on the electrode surface. Consequently, these disadvantage of amperometric enzyme sensor principle are the inherent reason for the limitation of current CGM stability and accuracy.We are currently engaged in the development of open circuit potential (OCP) based enzyme glucose sensing principle, which can solve the current issues in the glucose sensors based on amperometric principle. We previously reported OCP based glucose sensor employing direct electron transfer type FAD-dependent glucose dehydrogenase complex (FADGDH) derived from Burkholderia cepacia 1-5. By combining direct electron transfer type FADGDH with OCP principle, the sensor signal could be obtained without being affected by ascorbic acid and acetaminophen. In this study, we demonstrated a long-term operation, over 3 months, using OCP based CGM sensor employing direct electron transfer type FADGDH. Methods Recombinantly prepared bacterial FADGDH, capable of direct electron transfer, was used in this study, and the enzyme was immobilized onto gold electrode by self-assembled monolayer. OCP of the enzyme electrode was monitored continuously toward the sample containing various concentration of glucose, against Ag/AgCl reference electrode. We calculated the ∆OCP as difference between the OCP value at each glucose concentration and the OCP value in presence of 0.1 mM glucose, and then calibration curve was prepared based on plotting ∆OCP versus glucose concentration. Continuous operation was carried out in 100 mM phosphate buffer (pH 7.0) including 20 mM glucose for 108 days. Results and Discussion ∆OCP increased depending on glucose concentration (0.1 mM~20 mM) without being affected by ascorbic acid and acetaminophen which are representative interference of glucose sensor as we previously observed. This sensor could be operated continuously for 108 days without showing any significant decrease on the slope of calibration curve. Since there were no current flow during open circuit state, the turnover number of enzyme reaction could be significantly minimized. Consequently, stable OCP signal could be obtained for 108 days without significant decrease of OCP signal. These results revealed that OCP principle could prevent enzyme inactivation during continuous operation, thereby lead to improve long-term stability of sensor. This result indicated that the OCP based glucose sensor employing direct electron transfer type FADGDH could measure glucose concentration continuously, accurately and stably over 3 months, and this long-term stability could be achieved by employing OCP measurement principle.

Oxygen insensitive amperometric glucose biosensor based on FAD dependent glucose dehydrogenase co-entrapped with DCPIP or DCNQ in a polydopamine layer

Continuous glucose monitoring devices (CGM) are the present and the future for the challenging task of diabetes management. With a yearly increase of patients worldwide, glucose biosensors with high accuracy and low interference effect become crucial and should be developed. Here we show the development of an oxygen insensitive glucose biosensor which comprises FAD-dependent glucose dehydrogenase, dichlorophenol indophenol (DCPIP) or 2,3-dichloro-naphthoquinone (DCNQ) as redox mediators and polydopamine as a constraining layer. The sensors were fully characterized and the effect of interferences on glucose-sensing was analyzed. We further show long term bioelectrocatalytic activity for at least 3 days without any additional coating or treatment. The system shows great reproducibility via an easy fabrication methodology that should allow low-cost manufacturing. Those encouraging results show great promise for future sensor development of enzyme-based bioelectronics devices.

(Invited) Electroactive Redox Polymers, Redox Glyconanoparticles and Supramolecular Assemblies Based on Nanotubes for Bioelectrochemical Applications

For four decades, the development of biointerfaces has been the subject of increasing research efforts in the field of analytical chemistry and energy conversion. In particular, the functionalization of electrodes by biomaterials based on electrogenerated polymers and / or carbon nanotubes, is widely used for the design of biosensors and biofuel cells. Nanotube deposits were successfully functionalized by electropolymerization or π−π stacking interactions with pyrene derivatives exhibiting both affinity or covalent binding interactions towards redox molecules or proteins. Some new approaches for developing nanostructured biomaterials based on functionalized tungsten disulfide nanotubes and self-supporting film of carbon nanotubes (buckypapers) will be illustrated with enzymes as biosensing element [1]. In particular, WS2 nanotubes functionalized with carboxylic acid functions were used for the tyrosinase immobilization in order to form an amperometric biosensor towards catechol and dopamine. Moreover, heteropolyoxometalates (C4H10N)6[P2Mo18O62] and (C6H8NO)4[H2P2W18O62] were immobilized within carbon nanotube matrix and used as mediators for the electron transfer with FAD dependent glucose dehydrogenase [2]. The glucose detection was performed with a sensitivity of 198 mAmolL-1cm-2, and a good reproducibility. A new generation of flexible buckypaper electrodes was produced by using linear polynorbornene polymers containing multiple pyrene groups as crosslinker [3]. In addition, the use of bifunctionalized polymers (pyrene and NHS groups) leads to robust buckypapers with the covalent binding of redox groups or enzymes [4].We have been also interested in the development and characterization of glyconanoparticles (vesicles, micelle aggregates) obtained from the self-assembly of block copolymers composed of polystyrene as a hydrophobic tail and cyclodextrin as a hydrophilic part. These glyconanoparticles, which are stable in suspension in aqueous media, have an outer layer composed of cyclodextrin which allows a post-functionalization of the nanoparticle by hydrophobic molecules through host-guest interactions. These nanoparticles are thus a versatile platform for fixing a wide range of redox mediators. Two types of redox nanoparticles in solution for the electrical connection of enzymes have been developed: nanoparticle modified by 9,10-phenanthrenequinone and nanoparticle modified by ABTS modified by two pyrene groups. This innovative approach will be applied to the electrical wiring of enzymes in solution for the elaboration of solubilized enzymatic fuel cell [5,6]. Acknowledgements The author wishes to acknowledge the support from the Sino-French international research network GDRI 0876 “New nanostructured materials and biomaterials for renewable electrical energy sources” for providing facilities. This work is partly supported by the French National Research Agency (ANR-18-CE09-0022-01).[1]

(Invited) Photo-Induced Signal Chains at Enzyme Electrodes

During the last decade photoelectrochemical devices which couple biochemical reactions to light-sensitive electrodes have gained increasing research interest. These kind of light-triggered systems have the inherent advantage that an additional tool - the light - can be used to control the reactions at a sensing electrode. This allows a light-directed read-out, but can also be used to change the energetic situation on the surface resulting in advantageous potential behavior [1].Enzymes as biocatalysts provide the advantage of a catalytic analyte conversion and specificity in this reaction. In order to couple them to light-sensitive electrodes different approaches have used.Besides the use of co-substrates or products [2], we have been focusing on the use of mediators for this interaction [3] or the establishment of a direct electron transfer of the enzyme to the light- sensitive entity [4]. Here we report on the use of two different types of polymers for establishing the electrical contact.In order to exploit the advantages of a high surface area we have constructed 3D electrodes which can be rather easily prepared by a template approach combined with spin coating as deposition method..Thus, the thickness of the electrode structure can be adjusted in a defined way [5,6].In a first approach 3D TiO2 of inverse opal character has been sensitized by a conducting polymer – sulfonated polyaniline. The polymer has here two functions: It ensures the light sensitivity in the visible range and also provides an interface at which the enzyme can exchange electrons. Advantageously the current signal can be gained at rather low potential. Furthermore, the response can be tuned by the dimensions of the 3D electrode demonstrating the scalability of the approach.As a further advancement we have used a redox polymer in order to couple FAD-dependent glucose dehydrogenase to the electrode. Here PbS QDs have been generated directly onto a 3D TiO2 electrode and characterized by TEM and photoelectrochemistry. The system allows the generation of a defined anodic photocurrent, which can be significantly enhanced by the biocatalytic conversion of glucose. Here the substrate dependent current signal starts at about -054V vs Ag/AgCl.Such electrode systems can find application in light-triggered sensing, but also for bioenergetics since the photocatalytic process can be “feeded” by electrons from the biocatalytic conversion.

Direct electron transfer-type bioelectrocatalysis of FAD-dependent glucose dehydrogenase using porous gold electrodes and enzymatically implanted platinum nanoclusters

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) was carried out using porous gold (Au) electrodes and enzymatically implanted platinum nanoclusters (PtNCs). The porous Au electrodes were prepared by anodization of planar Au electrodes in a phosphate buffer containing glucose as a reductant. Moreover, PtNCs were generated into AtGDH by an enzymatic reduction of hexachloroplatinate (IV) ion. The modification was confirmed by native polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. The AtGDH-adsorbed porous Au electrode showed a DET-type bioelectrocatalytic wave both in the presence and absence of PtNCs; however, the current density with PtNCs (~1 mA cm−2 at 0 V vs. Ag|AgCl|sat. KCl) was considerably higher than that without PtNCs. The kinetic and thermodynamic analysis of the steady-state catalytic wave indicated that inner PtNCs shortened the distance between the catalytic center of AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.

Electron transfer from FAD-dependent glucose dehydrogenase to single-sheet graphene electrodes

Continuous glucose monitoring (CGM) is an emerging technology that can provide a more complete picture of the diabetes patient’s glucose levels. Amperometric blood glucose tests typically require redox mediators to facilitate charge transfer from the enzyme to the electrode, that are not ideal in CGM settings because of their potential toxicity or long-term stability issues. Direct electron transfer (DET) would eliminate this need and has therefore attracted substantial interest. However, most DET-based glucose biosensor studies so far have used glucose oxidase (GOx) leading to controversial results because the oxygen dependency may be misinterpreted as DET. Here, we overcome this challenge by using an oxygen-insensitive glucose dehydrogenase (GDH). To enable direct electron transfer, the enzyme was immobilized on the surface of high-quality single-layer graphene electrodes via short pyrene linkers (<1 nm). The biosensor strongly responded to glucose even without a redox mediator, implying direct electron transfer. Control measurements on different surfaces further confirm that the response is enzyme-specific. The activity of immobilized enzymes was confirmed by glucose measurements with a conventional ferrocenemethanol mediator as well as relatively unexplored redox mediator - nitrosoaniline. The influence of a most potent interferent in blood, ascorbic acid, was assessed. This is the first demonstration of application of single-layer graphene electrode to obtain DET from an oxygen insensitive enzyme (GDH), highlighting the potential of such devices for applications in CGM.