Bilirubin Oxidase Research Articles

Conductance Channels in a Single-Entity Enzyme.

For a long time, the prevailing view in the scientific community was that proteins, being complex macromolecules composed of amino acid chains linked by peptide bonds, adopt folded structure with insulating or semiconducting properties, with high bandgaps. However, recent discoveries of unexpectedly high conductance levels, reaching values in the range of dozens of nanosiemens (nS) in proteins, have challenged this conventional understanding. In this study, we used scanning tunneling microscopy (STM) to explore the single-entity conductance properties of enzymatic channels, focusing on bilirubin oxidase (BOD) as a model metalloprotein. By immobilizing BOD on a conductive carbon surface, we discern its preferred orientation, facilitating the formation of electronic and ionic channels. These channels show efficient electron transport (ETp), with apparent conductance up to the 15 nS range. Notably, these conductance pathways are localized, minimizing electron transport barriers due to solvents and ions, underscoring BOD's redox versatility. Furthermore, electron transfer (ET) within the BOD occurs via preferential pathways. The alignment of the conductance channels with hydrophilicity maps, molecular vacancies, and regions accessible to electrolytes explains the observed conductance values. Additionally, BOD exhibits redox activity, with its active center playing a critical role in the ETp process. These findings significantly advance our understanding of the intricate mechanisms that govern ETp processes in proteins, offering new insights into the conductance of metalloproteins.

Unusual Long-Term Stability of Enzymatic Bioelectrocatalysis in Organic Solvents

Organic solvent tolerant enzymes are of tremendous importance for the pharmaceutical industry in search of high added-value chemicals. However, enzymes exhibit usually weak long-term activity and stability in various media. Here, we report a general strategy allowing stabilizing and keeping enzymes active in biphasic systems upon immobilization on an electrode surface. It consists in the combination of an engineered solvent-tolerant enzyme, combined with fine-tuned osmium-based redox polymers where the concentration of osmium complex has been specifically tuned to minimize its deswelling associated with the use of porous gold electrodes. This approach is validated with bilirubin oxidase as a model system. This copper enzyme is able to oxidize a wide range of substrates, combined with the reduction of O2 to water. While all other enzymatic systems irreversibly lose their activity and stability in the presence of 7.5 M methanol or below, this optimized enzymatic system stays functional in 12.5 M methanol with no loss in current density. It exhibits a half-life of more than 8 days, which is unprecedented in the literature. We show that this electrode can also operate in DMSO, dioxane, acetonitrile and acetone, thus opening up a very wide variety of applications in the field of bioelectrocatalysis and bioelectrosynthesis. Figure: Example of a long-term stability experiment for a mediated bilirubin oxidase biocathode in 12.5 M methanol. Black curve: immobilized on a bare gold electrode; red curve: immobilized on a 11/2 layers porous gold electrode. Experiments performed at +0.1V vs. Ag/AgCl. The total loading was fixed at 600 µg.cm-2 for both experiments. Figure 1

Biofuel cell-based self-powered immunosensor for detection of 17β-estradiol by integrating the target-induced biofuel release and biogate immunoassay.

A novel biofuel cell (BFC)-based self-powered electrochemical immunosensing platform was developed by integrating the target-induced biofuel release and biogate immunoassay for ultrasensitive 17β-estradiol (E2) detection. The carbon nanocages/gold nanoparticle composite was employed in the BFCs device as the electrode material, through which bilirubin oxidase and glucose oxidase were wired to form the biocathode and bioanode, respectively. Positively charged mesoporous silica nanoparticles (PMSN) were encapsulated with glucose molecules as biofuel and subsequently coated by the negatively charged AuNPs-labelled anti-E2 antibody (AuNPs-Ab) serving as a biogate. The biogate could be opened efficiently and the trapped glucose released once the target E2 was recognized and captured by AuNPs-Ab due to the decreased adhesion between the antigen-antibody complex and PMSN. Then, glucose oxidase oxidized the glucose to produce a large number of electrons, resulting in significantly increased open-circuit voltage (EOCV). Promisingly, the proposed BFC-based self-powered immunosensor demonstrated exceptional sensitivity for the detection of E2 in the concentration range from 1.0pgmL-1 to 10.0ngmL -1, with a detection limit of 0.32pgmL-1 (S/N = 3). Furthermore, the prepared BFC-based self-powered homogeneous immunosensor showed significant potential for implementation as a viable prototype for a mobile and an on-site bioassay system in food and environmental safety applications.

Enzymatic biofuel cells-based self-powered electrochemical biosensor for detection of 17β-estradiol based on dual-enzyme cascade amplification and bioconjugate

Self-powered electrochemical biosensors (SPEB) based on enzymatic biofuel cells (EBFC) have garnered significant attention due to their distinctive characteristic of not necessitating the source of external power supply. This study developed a novel EBFC-based SPEB by incorporating dual-enzyme cascade amplification and bioconjugate for extremely precise detection of 17β-estradiol (E2). The carbon nanocages/gold nanoparticles composite was employed as substrate to immobilize bilirubin oxidase (BOD) and complementary ssDNA (csDNA) to form bioconjugate. The carbon nanotubes/gold nanoparticles composite was used in the EBFC device as the electrode material, through which glucose oxidase (GOD) and E2 aptamer were wired to form the bioanode and biocathode, respectively. The biocathode further linked with bioconjugate via a hybridization reaction between E2 aptamer and csDNA. The bioconjugate integrates the recognition and signal probes for homogeneous electrochemical assay of targets, which released from biocathode once the target E2 was recognized and hindered the transfer of electrons produced by GOD on bioanode. Thus, the open-circuit voltage (EOCV) of the EBFC-based SPEB dramatically decreased, exhibiting exceptional sensitivity for the detection of E2 over a concentration range from 10 pg mL−1 to 20 ng mL−1, with a detection limit of 3.3 pg mL−1 (S/N = 3). Furthermore, the prepared EBFC-based SPEB showed significant potential for implementation as a viable prototype for a mobile and an on-site bioassay system in food and environmental safety applications.

Harnessing Redox Polymer Dynamics for Enhanced Glucose-Oxygen Coupling in Dual Biosensing and Therapeutic Applications.

The burgeoning field of continuous glucose monitoring (CGM) for diabetes management faces significant challenges, particularly in achieving precise and stable biosensor performance under changing environmental conditions such as varying glucose concentrations and O2 levels. To address this, we present a novel biosensor based on the electroless coupling of glucose oxidation catalyzed by flavin-dependent glucose dehydrogenase (FAD-GDH) and O2 reduction catalyzed by bilirubin oxidase (BOD) via a redox polymer, dimethylferrocene-modified linear poly(ethylenimine), FcMe2-LPEI. Initial cyclic voltammetry tests confirm the colocalization of both enzymatic reactions within the potential range of the polymer, indicating an effective electron shuttle mechanism. As a result, we created a hybrid biosensor that operates at open-circuit potential (OCP). It can detect glucose concentrations of up to 100 mM under various O2 conditions, including ambient air. This resulted from optimizing the enzyme ratio to 120 ± 10 mUBOD·UFAD-GDH-1·atmO2-1. This biosensor is highly sensitive, a crucial feature for CGM applications. This distinguishes it from FAD-GDH traditional biosensors, which require a potential to be applied to measure glucose concentrations up to 30 mM. In addition, this biosensor demonstrates the ability to function as a noninvasive, external device that can adapt to changing glucose levels, paving the way for its use in diabetes care and, potentially, personalized healthcare devices. Furthermore, by leveraging the altered metabolic pathways in tumor cells, this system architecture opened up new avenues for targeted glucose scavenging and O2 reduction in cancer therapy.

Bimodal 3D DNA nanomachines coupling with bioconjugates powered electrochemical and visual-driven dual-enzyme cascade sensing of Let-7a

A dual-enzyme self-powered biosensor utilizing bimodal 3D DNA nanomachines and gold nanoparticle (AuNPs)/carbon/disulfide molybdenum hollow nanorods bioconjugates is developed for the electrochemical and visual detection of the tumor biomarker let-7a. DNA probes are immobilized on a flexible electrode substrate of AuNPs/graphdiyne/carbon cloth to improve probe loading capacity and electron transfer efficiency. The presence of let-7a initiates the work of dual-enzyme 3D DNA nanomachine, and the output DNA single strand cleaved by Nb.BbvCI is served as a bridge to connect glucose oxidase (GOD)-DNA conjugate. The GOD coordinates with the cathodic enzyme bilirubin oxidase and produces an increased open circuit voltage signal, which is attributed to the hexaammine ruthenium in the double-stranded adsorption system of the biocathode. By utilizing the H2O2 generated during the oxidation process of GOD, a linear relationship between the color of 3,3′,5,5′-tetramethylbenzidine and the concentration of let-7a is established, enhancing the reliability of the detection results through the independent signal conversion mechanisms of electrochemical/colorimetric modes. The results demonstrate a wide dynamic range of 0.0001–0.1 pM with detection limits of 31.04 aM (S/N=3) in electrochemical mode and 59.99 aM (S/N=3) in the colorimetric mode. This dual-mode biosensing strategy can be expanded to detect additional nucleic acid biomarkers, providing a promising support and reference for biological analysis and medical diagnosis.

Flexible and stretchable high performance enzymatic biofuel cells implantable in tube-type artificial blood vessel kit

Enzymatic biofuel cells (EBFCs) are emerged as promising power sources for implantable devices, offering excellent form factor and performance. For these applications, EBFCs require customer tailored structure and more than 10 μW power. In this study, flexible and stretchable EBFCs are suggested, assessing their performance using tube-type artificial blood vessel (TABV) cell kit with sheep blood fuel. To fabricate the EBFCs, buckypaper (BP) is molded to polydimethylsiloxane (PDMS) to fabricate BP@PDMS electrode that shows excellent strain rate (95 %) and durability (1500 cycles) in tensile and fatigue tests. Additionally, electrochemical evaluations are implemented to measure the performance of anode and cathode fabricated with BP@PDMS. In anode including 1,10-phenanthroline-5,6-dione and glucose dehydrogenase, maximum reactivity of glucose oxidation is 1.05 mA/cm2, while in cathode including bilirubin oxidase, that of oxygen reduction is 0.61 mA/cm2. Regarding flexibility, anode and cathode are little affected by current density irrespective of bending angle, proving that the fabricated anode and cathode have good flexibility. To emulate the behavior of EBFCs in actual blood vessel, EBFCs are implanted in TABV cell kit, fueling sheep blood. Observed open circuit voltage of 0.59 V and maximum power of 26 μW reveals that fabricated EBFCs have superior potential to be considered as power sources for implantable devices.

Construction of a Conductive Polymer/AuNP/Cyanobacteria-Based Biophotovoltaic Cell Harnessing Solar Energy to Generate Electricity via Photosynthesis and Its Usage as a Photoelectrochemical Pesticide Biosensor: Atrazine as a Case Study.

In this research, a cyanobacteria (Leptolyngbia sp.)-based biological photovoltaic cell (BPV) was designed. This clean energy-friendly BPV produced a photocurrent as a result of illuminating the photoanode and cathode electrodes immersed in the aqueous medium with solar energy. For this purpose, both electrodes were first coated with conductive polymers with aniline functional groups on the gold electrodes. In the cell, the photoanode was first coated with a gold-modified poly 4-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzamine polymer, P(SNS-Aniline). Thioaniline-functionalized gold nanoparticles were used to provide a cross-link formation with bis-aniline conductive bonds with the conductive polymer using electrochemical techniques. Leptolyngbia sp., one of the cyanobacteria that can convert light energy into chemical energy, was attached to this layered electrode surface. The cathode of the cell was attached to the gold electrode surface with P(SNS-Aniline). Then, the bilirubin oxidase (BOx) enzyme was immobilized on this film surface with glutaraldehyde activation. This cell, which can use light, thanks to cyanobacteria, oxidized and split water, and oxygen was obtained at the photoanode electrode. At the cathode electrode, the oxygen gas was reduced to water by the bioelectrocatalytic method. To obtain a high photocurrent from the BPV, necessary optimizations were made during the design of the system to increase electron transport and strengthen its transfer. While the photocurrent value obtained with the designed BPV in optimum conditions and in the pseudosteady state was 10 mA/m2, the maximum power value obtained was 46.5 mW/m2. In addition to storing the light energy of the system, studies have been carried out on this system as a pesticide biosensor. Atrazine biosensing via the BPV system was analytically characterized between 0.1 and 1.2 μM concentrations for atrazine, and a very low detection limit was found as 0.024 μM. In addition, response time and recovery studies related to pesticide biosensor properties of the BPV were also investigated.

Design of a Novel Green Algae‐Based Biological Photovoltaic Cell with High Photocurrent and a Photoelectrochemical Biosensing Approach Utilizing the BPV for Pesticide Analysis in Water

AbstractIn this research, a green alga (Paulschulziapseudovolvox sp.) based biological photovoltaic cell (BPV) was designed. This clean energy‐friendly BPV produced photocurrent as a result of illuminating the photoanode and cathode electrodes immersed in the aqueous medium with solar energy. For this purpose, both electrodes were first coated with conductive polymers with aniline functional groups on gold electrodes. In the cell, the photoanode was first coated with a gold‐modified poly 4‐(2,5‐di(thiophen‐2‐yl)‐1H‐pyrrol‐1‐yl)benzamine polymer, (P(SNS‐NH2)). Cytochrome C (Cyt. C) material was used to provide crosslink formation with bis‐aniline covalent bonds with the conductive polymer using electrochemical techniques. Paulschulziapseudovolvox sp., one of the green algae that can convert light energy into chemical energy, is attached to this layered electrode surface. The cathode of the cell is attached to the gold electrode surface with poly 4‐(4HDithieno[3,2‐b : 2′,3′‐d]pyrrole‐4‐yl)aniline (P(DTP‐Aryl‐Amine)). Then, the bilirubin oxidase enzyme was immobilized on this film surface with glutaraldehyde activation. This cell, which can use light thanks to green algae, oxidizes and splits water, and oxygen is obtained at the photoanode electrode. At the cathode electrode, the oxygen gas is reduced to water by the bio‐electro‐catalytic method. To obtain high photocurrent from the BPV, necessary electrochemical and chemical optimizations were made during the design of the system to increase the amount of electrons that were transferred and fasten its transfer rate. While the photocurrent value generated by the designed BPV in optimum conditions and in the pseudo‐steady state is 10 mA/m2, the maximum power value obtained is 46.5 mW/m2. In addition to the production of the green algae‐based BPV generating highly efficient electricity which is the main of target of this study, some studies have also been carried out to show whether this system can be used as a pesticide biosensor. Atrazine and diuron biosensing via the BPV system was analytically characterized and recovery and interference studies related to pesticide biosensor property of the BPV were also investigated.

Emerging Trends of Bilirubin Oxidases at the Bioelectrochemical Interface: Paving the Way for Self-Powered Electrochemical Devices and Biosensors.

Bilirubin oxidases (BODs) [EC 1.3.3.5 - bilirubin: oxygen oxido-reductase] are enzymes that belong to the multicopper oxidase family and can oxidize bilirubin, diphenols, and aryl amines and reduce the oxygen by direct four-electron transfer from the electrode with almost no electrochemical overpotential. Therefore, BOD is a promising bioelectrocatalyst for (self-powered) biosensors and/or enzymatic fuel cells. The advantages of electrochemically active BOD enzymes include selective biosensing, biocatalysis for efficient energy conversion, and electrosynthesis. Owing to the rise in publications and patents, as well as the expanding interest in BODs for a range of physiological conditions, this Review analyzes scientific literature reports on BOD enzymes and current hypotheses on their bioelectrocatalysis. This Review evaluates the specific research outcomes of the BOD in enzyme (protein) engineering, immobilization strategies, and challenges along with their bioelectrochemical properties, limitations, and applications in the fields of (i) biosensors, (ii) self-powered biosensors, and (iii) biofuel cells for powering bioelectronics.

Self-powered biosensing platform for Highly sensitive detection of soluble CD44 protein

In this study, a self-powered biosensor based on an enzymatic biofuel cell was proposed for the first time for the ultrasensitive detection of soluble CD44 protein. The as-prepared biosensor was composed of the co-exist aptamer and glucose oxidase bioanode and bilirubin oxidase modified biocathode. Initially, the electron transfer from bioanode to biocathode was hindered due to the presence of the aptamer with high insulation, generating a low open-circuit voltage (EOCV). Once the target CD44 protein was present, it was recognized and captured by the aptamer at the bioanode, thus the interaction between the target CD44 protein and the immobilized aptamer caused the structural change at the surface of the electrode, which facilitated the transfer of electrons. The EOCV showed a good linear relationship with the logarithm of the CD44 protein concentrations in the range of 0.5–1000 ng mL−1 and the detection limit was 0.052 ng mL−1 (S/N = 3). The sensing platform showed excellent anti-interference performance and outstanding stability that maintained over 97% of original EOCV after 15 days. In addition, the relative standard deviation (1.40–1.96%) and recovery (100.23–101.31%) obtained from detecting CD44 protein in real-life blood samples without special pre-treatment indicated that the constructed biosensor had great potential for early cancer diagnosis.

Pseudocapacitance behavior enables efficient and stable electrochemical energy conversion and storage in glucose/air enzymatic biofuel cells

Constructing an integrated system with high-efficiency and stable energy conversion and storage (ECS) is of great significance but remains challenges. Herein, we report a biodevice consisting of a bioanode of tetrathiafulvalene/glucose oxidase (TTF/GOx) immobilized on asphalt-derived porous carbon (APC) for catalyzing glucose oxidation and a biocathode of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate)/bilirubin oxidase (ABTS/BOD) immobilized on APC for catalyzing oxygen reduction, to achieve ECS with high-efficiency and excellent stability. The redox mediators, TTF and ABTS, used at the bioanode and biocathode, respectively, simultaneously mediate electron transfer to release electric current on discharging and serve as pseudocapacitors to achieve charge storage at open-circuit conditions, enabling high power density and self-charge capability. The biodevice of the above configuration delivers a peak power output of 5.01 mW cm−2 in the pulse discharge mode with cell voltage remaining 93.7 % of its initial value after 100 pulse discharge/self-charge cycles. This work provides a highly efficient solution to address the low power density and poor stability of the conventional enzymatic biofuel cells, showing great implications for small, implantable and wearable electronics.

High voltage flexible glucose/O2 fully printed hydrogel-based enzymatic fuel cell

Herein we report on a novel enzymatic fuel cell (EFC) based on stencil printed electrodes modified with pyrrolo quinoline quinone glucose dehydrogenase and bilirubin oxidase, which are assembled by considering two different configurations: (i) normal assembling in liquid electrolyte and (ii) six EFCs connected in series, each one comprising both bioanode and biocathode, coupled through a hydrogel-based electrolyte in a stack-like mode similar to a Voltaic pile. After a deep electrodes characterization, they are assembled according to the first configuration obtaining an open circuit voltage (OCV) of 0.562 ± 0.002 V. Moreover, the EFC performance are substantially improved by using the second configuration (six EFCs connected in series) obtaining an OCV of 2.36 ± 0.22 V with a maximum power output of 22.9 ± 0.9 μW at a cell voltage of 1.95 V (operating in 10 mM D-glucose). This innovative approach represents a proof-of-concept towards the development of renewable power sources and could serve as a critical step in powering implantable bioelectronics, such as pacemakers.

Effect of pore size of MgO-templated porous carbon electrode on immobilized crosslinked enzyme–mediator redox network

Magnesium oxide templated porous carbon (MgOC) is a promising material as an enzyme electrode platform. Herein, a flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and thionine were immobilized using a crosslinker, and the applicability of MgOC with various pore sizes as a platform was investigated. Among the pores of MgOC, the novel designed FAD-GDH/thionine network generated a 1.1 mA cm−2 catalytic current at 100 nm pore size, demonstrating a significantly improved glucose supply and a thin redox composite layer was formed within the nanostructured material. At low loading, the catalytic current is independent of the pore size; however, at high loading, a varied catalytic current is achieved for the different MgOC pore sizes. Further, the stability was improved of porous platform compared to the glassy carbon (GC) electrode. Additionally, a glucose/O2 biofuel cell is constructed with FAD-GDH/thionine on porous MgOC and non-porous GC as an anode, and mediated bilirubin oxidase as a cathode. A remarkably high electricity generation efficiency (0.23 mW cm−2 μg−1 of FAD-GDH) is shown on a porous electrode platform than non-porous and a previously reported immobilized FAD-GDH-based anode. These results demonstrate the potential of porous MgOC as an anode for a novel FAD-GDH/thionine network toward electricity generation.

Investigating Enzyme-Based Electrode Processes Using Electrochemical Impedance Spectroscopy and Distribution of Relaxation Time Analysis

Enzymatic fuel cells (EFCs) are an emerging class of electrochemical devices that have the potential to harness renewable energy sources by converting the chemical energy of an organic substrate into electrical energy. Enzymes are responsible in an EFC for highly selective and efficient electrode processes under typically mild ambient conditions, such as the oxidation reaction at the anode and the reduction reaction at the cathode 1. In addition to more conventional techniques, electrochemical impedance spectroscopy (EIS) can be employed to characterize such systems. Electrochemical behavior of enzymatic electrodes, such as enzyme reaction kinetics, substrate mass transport, and electrode stability, can be studied by measuring the system's impedance over a wide frequency range and then performing a precise post-process analysis based on the distribution of relaxation time (DRT). DRT analysis is rarely used to interpret EIS spectra within the EFCs research field, despite the fact that it is widely acknowledged as relevant for properly understanding the EIS potential to improve power density in fuel cells 2,3. Glucose oxidase (GOx) and bilirubin oxidase (BOD) were immobilized on multi-walled carbon nanotubes (MWCNTs) to catalyze the bioelectrocatalytic oxidation of glucose at the anode and the bioelectrocatalytic reduction of oxygen at the cathode, respectively. This study presents a promising approach for the investigation of enzyme-based anodic and cathodic processes by measuring them with EIS and then interpreting the results with DRT analysis, as depicted in Figure 1. By varying different operating parameters, such as the enzymatic loading on the electrode surface or electrolyte conditions, this method can be used to identify three primary relaxation processes occurring at both electrodes. At the interface between the electrolyte and the electrode, it is possible to conclude that high-frequency processes are accountable for ionic conduction, intermediate-frequency processes are accountable for charge transfer, and low-frequency processes are accountable for oxygen surface exchange and diffusion.This method provides access to preliminary fundamental information about enzymatic bioelectrode processes, which can be used to optimize biofuel cell construction and compositions and increase their power output. Figure 1. Schematic representation of the characterization method used to characterize a GOx-CNT-modified anode consisting of EIS measurements and subsequent DRT analysis. Concentration of glucose 175 mM in 0.1 M HEPES Buffer, pH 7; EIS parameters: 20 mV sinusoidal excitation, 0.1 Hz-100 kHz. References K. Herkendell, A. Stemmer and R. Tel-Vered, Nano Res., 12(4), 767–775 (2019). H. Wang, X. Long, Y. Sun, D. Wang, Z. Wang, H. Meng, C. Jiang, W. Dong and N. Lu, Frontiers in microbiology, 13, 973501 (2022). F. Ciucci, Current Opinion in Electrochemistry, 13, 132–139 (2019). Figure 1

Single-Entity Bioelectrochemistry

Interfacing biological catalytic entities, such as whole cells or enzymes, with electrodes, has a wide range of applications, ranging from fundamental studies of the entities themselves to power generation, bioremediation, chemical synthesis, and biosensing. In all cases, the operation of these bioelectrocatalytic systems is based on electron transfer phenomena within protein molecules, whether isolated or residing inside cells or parts of cells, as well as between proteins and electronics. Typically, the electron transfer and catalytic properties of proteins are studied using ensemble averaging methods, which obscure any heterogeneity and dynamics of individual molecules. This information, however, is especially important for understanding electron transfer and catalytic processes within a living cell where only one or a few protein molecules are present. While single-entity electrochemical measurements are particularly useful for studying electron transfer reactions at the nanoscale level, they have limited applications for investigating biological entities due to the experimental challenges associated with direct measurements of small electrical currents. 1,2 Here we will discuss our recent progress in investigating biological electron transfer reactions at the single-entity level, from bacterial cells to single redox proteins, using nano-impact (collision) single-entity electrochemistry. 3 We demonstrate how advances in electrode fabrication methods and detection setups enable current detection in the fA range. When combined with the developed data processing algorithm based on unsupervised learning and template matching techniques, this enabled unprecedented insight into electron transfer processes at the nanoscale. We will talk about insights provided by the method in the study of several important bioelectrocatalytic systems: single E.coli cells with overexpressed Fe-Fe hydrogenases, single bilirubin oxidase molecules, and single catalase molecules. In addition, we will discuss models that we developed for understanding the single bioentity electrode interactions for diffusion-controlled enzymes when the product of the catalytic reaction is detected on the electrode, as well as systems based on direct electron transfer between the bioentity and the electrode. Overall, we show how single-entity bioelectrochemistry can shed light on biological electron transfer and catalysis processes and discuss the challenges it currently faces. Zhang, J.-H.; Zhou, Y.-G. Nano-Impact Electrochemistry: Analysis of Single Bioentities. TrAC Trends Anal. Chem. 2019, 115768.Davis, C.; Wang, S. X.; Sepunaru, L. What Can Electrochemistry Tell Us About Individual Enzymes? Curr. Opin. Electrochem. 2020, 100643.Sekretaryova, A. N.; Vagin, M. Yu.; Turner, A. P. F.; Eriksson, M. Electrocatalytic Currents from Single Enzyme Molecules. J. Am. Chem. Soc. 2016, 138 (8), 2504–250

Interaction between bilirubin oxidase and Au nanoparticles distributed over dimpled titanium foil towards oxygen reduction reaction

Spatial distribution of enzymes on electrodes is a key point to understand and optimize bioelectrocatalysis, although not largely investigated. In this work, Ti nanodimples serve as a platform for getting a controlled spatial distribution of gold nanoparticles (AuNPs). By varying the thickness of the initial gold layer, AuNPs with average sizes ranging from 13±6, 46±12 to 81±13 nm are obtained. AFM and TEM allow to localize the AuNPs in the dimples. It is in particular highlighted that the smallest NPs are present not only at the bottom but also on the edges of the Ti dimple, with TiO2 layer in their close surrounding. Enzymatic O2 reduction by immobilized Myrothecium verrucaria bilirubin oxidase (Mv BOD) is then investigated on the different materials. After having demonstrated the occurrence of the bioelectrocatalytic reaction on AuNPs excluding the Ti surface, the efficiency of the catalysis is discussed as a function of the AuNP size. The shape and fitting of the cyclic voltammetry curves obtained with the smallest AuNPs suggest a decrease in the electron transfer rate as a consequence of the colocalization of AuNPs and semiconducting TiO2. Interestingly, high stability of the enzymatic current is obtained with the highest AuNPs even in the presence of high concentration of salts.

Sustainably Sourced Mesoporous Carbon Molecular Sieves as Immobilization Matrices for Enzymatic Biofuel Cell Applications

Ordered mesoporous carbon CMK-3 sieves with a hexagonal structure and uniform pore size have recently emerged as promising materials for applications as adsorbents and electrodes. In this study, using sucrose as the sustainable carbon source and SBA-15 as a template, CMK-3 sieves are synthesized to form bioelectrocatalytic immobilization matrices for enzymatic biofuel cell (EFC) electrodes. Their electrochemical performance, capacitive features, and the stability of enzyme immobilization are analyzed and compared to commercially available multi-walled carbon nanotubes (MWCNT) using cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The anodic reaction in the presence of glucose oxidase (GOx) and ferrocene methanol (FcMeOH) on the sustainably sourced CMK-3-based electrodes produces bioelectrocatalytic current responses at 0.5 V vs. saturated calomel electrode (SCE) that are twice as high as on the MWCNT-based electrodes under saturated glucose conditions. For the cathodic reaction, the MWCNT-based cathode performs marginally better than the CMK-3-based electrodes in the presence of bilirubin oxidase (BOD) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−). The CMK-3-based EFCs assembled from the GOx anode and BOD cathode results in a power output of 93 μW cm−2. In contrast, the output power of MWCNT-based EFCs is approximately 53 μW cm−2. The efficiency of CMK-3 as a support material for biofuel cell applications is effectively demonstrated.

Bi-enzymatic chemo-mechanical feedback loop for continuous self-sustained actuation of conducting polymers

Artificial actuators have been extensively studied due to their wide range of applications from soft robotics to biomedicine. Herein we introduce an autonomous bi-enzymatic system where reversible motion is triggered by the spontaneous oxidation and reduction of glucose and oxygen, respectively. This chemo-mechanical actuation is completely autonomous and does not require any external trigger to induce self-sustained motion. The device takes advantage of the asymmetric uptake and release of ions on the anisotropic surface of a conducting polymer strip, occurring during the operation of the enzymes glucose oxidase and bilirubin oxidase immobilized on its surface. Both enzymes are connected via a redox polymer at each extremity of the strip, but at the opposite faces of the polymer film. The time-asymmetric consumption of both fuels by the enzymatic reactions produces a double break of symmetry of the film, leading to autonomous actuation. An additional break of symmetry, introduced by the irreversible overoxidation of one extremity of the polymer film, leads to a crawling-type motion of the free-standing polymer film. These reactions occur in a virtually unlimited continuous loop, causing long-term autonomous actuation of the device.

Unusual long-term stability of enzymatic bioelectrocatalysis in organic solvents

Organic solvent tolerant enzymes are of tremendous importance for the pharmaceutical industry in search of high added-value chemicals. However, enzymes exhibit usually weak long-term activity and stability in various media. Here, we report a general strategy allowing stabilizing and keeping enzymes active in mixed-solvent systems upon immobilization on an electrode surface. It consists in the combination of an engineered solvent-tolerant enzyme, combined with fine-tuned osmium-based redox polymers where the concentration of osmium complex has been specifically tuned to minimize its deswelling, associated with the use of porous gold electrodes. This approach is validated with bilirubin oxidase as a model system. This copper enzyme is able to oxidize a wide range of substrates, combined with the reduction of O2 to water. While all other enzymatic systems irreversibly lose their activity and stability in the presence of 7.5 M methanol or below, this optimized enzymatic system stays functional in 12.5 M methanol with no loss in current density. It exhibits a half-life of more than 8 days, which is unprecedented in the literature. We show that this electrode can also operate in DMSO, dioxane, acetonitrile and acetone, thus opening up a very wide variety of applications in the field of bioelectrocatalysis and bioelectrosynthesis.