Direct Electron Transfer Type Research Articles

Engineering of Lactate Oxidase and Dehydrogenase for Development of Direct Electron Transfer Type Lactate Sensor and Application for Multiplexed Biosensor System

Engineering of Lactate Oxidase and Dehydrogenase for Development of Direct Electron Transfer Type Lactate Sensor and Application for Multiplexed Biosensor System

Direct Electron Transfer-Type Oxidoreductases for Biomedical Applications.

Among the various types of enzyme-based biosensors, sensors utilizing enzymes capable of direct electron transfer (DET) are recognized as the most ideal. However, only a limited number of redox enzymes are capable of DET with electrodes, that is, dehydrogenases harboring a subunit or domain that functions specifically to accept electrons from the redox cofactor of the catalytic site and transfer the electrons to the external electron acceptor. Such subunits or domains act as built-in mediators for electron transfer between enzymes and electrodes; consequently, such enzymes enable direct electron transfer to electrodes and are designated as DET-type enzymes. DET-type enzymes fall into several categories, including redox cofactors of catalytic reactions, built-in mediators for DET with electrodes and by their protein hierarchic structures, DET-type oxidoreductases with oligomeric structures harboring electron transfer subunits, and monomeric DET-type oxidoreductases harboring electron transfer domains. In this review, we cover the science of DET-type oxidoreductases and their biomedical applications. First, we introduce the structural biology and current understanding of DET-type enzyme reactions. Next, we describe recent technological developments based on DET-type enzymes for biomedical applications, such as biosensors and biochemical energy harvesting for self-powered medical devices. Finally, after discussing how to further engineer and create DET-type enzymes, we address the future prospects for DET-type enzymes in biomedical engineering.

Development of Direct Electron Transfer-Type Extended Gate Field Effect Transistor Enzymatic Sensors for Metabolite Detection.

In this work, direct electron transfer (DET)-type extended gate field effect transistor (EGFET) enzymatic sensors were developed by employing DET-type or quasi-DET-type enzymes to detect glucose or lactate in both 100 mM potassium phosphate buffer and artificial sweat. The system employed either a DET-type glucose dehydrogenase or a quasi-DET-type lactate oxidase, the latter of which was a mutant enzyme with suppressed oxidase activity and modified with amine-reactive phenazine ethosulfate. These enzymes were immobilized on the extended gate electrodes. Changes in the measured transistor drain current (ID) resulting from changes to the working electrode junction potential (φ) were observed as glucose and lactate concentrations were varied. Calibration curves were generated for both absolute measured ID and ΔID (normalized to a blank solution containing no substrate) to account for variations in enzyme immobilization and conjugation to the mediator and variations in reference electrode potential. This work resulted in a limit of detection of 53.9 μM (based on ID) for glucose and 2.12 mM (based on ID) for lactate, respectively. The DET-type and Quasi-DET-type EGFET enzymatic sensor was then modeled using the case of the lactate sensor as an equivalent circuit to validate the principle of sensor operation being driven through OCP changes caused by the substrate-enzyme interaction. The model showed slight deviation from collected empirical data with 7.3% error for the slope and 8.6% error for the y-intercept.

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound d-Fructose Dehydrogenase with Structural Bioelectrochemistry

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound <scp>d</scp>-Fructose Dehydrogenase with Structural Bioelectrochemistry

In silico simulations for prediction and modeling of protein engineering within catalytic subunit of direct electron transfer type FAD glucose dehydrogenase for increases in substrate specificity and structural stability.

The bacterial flavin adenine dinucleotide (FAD) glucose dehydrogenase (FADGDH) is composed of three subunits; 1) a catalytic subunit which belongs to the glucose-methanol-choline (GMC) oxidoreductase family contains an FAD cofactor and 3Fe-4S cluster in its redox center, 2) a hitchhiker protein subunit functioning in the bacterial TAT secretion system, and 3) a membrane-bound subunit with three heme c moieties, responsible for the transfer of electrons between the active-site cofactor and external electron acceptors. The presence of the electron transfer subunit makes this enzyme able to transfer electron directly to the electrode, thereby designates this enzyme as a direct electron transfer (DET) type GDH. The recent X-ray crystal structure elucidation of its catalytic subunit complexed with small subunit, revealed the unique substrate binding cavity of this enzyme. This enzyme was then mutated within its substrate pocket to increase substrate specificity towards glucose as a validation for later energy minimization and molecular dynamics simulations using AMBER MD. Modeling both the FAD and 3Fe-4S cluster elucidated a new methodology for modeling proteins involving covalent bonding of cofactors and namely metal ion cluster harboring proteins. From this modeling, the electron transfer pathway involving both cofactors and metal ion clusters can be further investigated in DET type enzymes. With these new modeling techniques, we can take a new approach in designing future mutational candidates by first evaluating them in silico then applying those results to our wet lab testing for higher throughput and a more targeted approach in designing and creating mutations.

Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme’s catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.

The development of micro–sized enzyme sensor based on direct electron transfer type open circuit potential sensing principle

One long term goal for biosensor development is towards implantable, long–term sensing for diagnostics, and monitoring of chronic health. The most common electrochemical sensors employ amperometric principles, although there is a fundamental challenge when trying to minimize the sensor size since the measurement current is directly proportional to the size of the working electrode. An alternative principle to amperometry for enzyme sensor is potentiometry, measuring open circuit potential (OCP). In this study, we demonstrate the size independency of an OCP based enzyme sensor focusing on direct electron transfer (DET) type– and quasi–DET–type oxidoreductases for, glucose and L–lactate, respectively. For glucose detection, we employed DET type flavin adenine dinucleotide glucose dehydrogenase, and the engineered L–lactate oxidase derived from Aerococcus viridans (AvLOx) conjugated with amine–reactive phenazine ethosulfate (arPES) was used as the quasi–DET type enzyme for the L–lactate detection. First, both enzymes were immobilized on 3 mm diameter gold disc electrode (GDE), and glucose and L–lactate were monitored based on OCP and transient potentiometric measurement, dOCP/dt. Then, L–lactate and glucose sensors were constructed using 10 µm GDEs, showing L–lactate and glucose concentration dependent OCP and dOCP/dt changes. The sensitivity was nearly equivalent for ΔOCP and dOCP/dt between the sensor using 3 mm and 10 µm GDE even though amperometric signal was entirely lost when measuring the arPES modified AvLOx on the 10 µm electrode. These results clearly demonstrate the size independency of an OCP based enzyme sensor which will be applied for the implantable and continuous monitoring system for metabolites in vivo.

Functionalization of Glucose Oxidase in Organic Solvent: Towards Direct Electrical Communication across Enzyme-Electrode Interface.

Enzymatic biosensors based on glucose oxidase has been proven to be one of the effective strategies for the detection of glucose and contributed to health improvements. Therefore, research and debates to date are ongoing in an attempt to find the most effective way to detect this analyte using this enzyme as the recognition center. The 3rd generation biosensors using direct electron transfer (DET) type enzymes are a great way towards practical devices. In this work, we developed a simple method for the functionalization of glucose oxidase with redoxable ferrocene groups in chloroform. The enzyme retained its activity after storage in this organic solvent and after the functionalization procedures. This enzyme functionalization strategy was employed to develop the biosensing monolayer-based platforms for the detection of glucose utilizing the quasi-DET mechanism. As a result of an electrochemical regeneration of the catalytic center, the formation of harmful H2O2 is minimized during enzymatic electrocatalysis.

In Vitro Continuous 3 Months Operation of Direct Electron Transfer Type Open Circuit Potential Based Glucose Sensor: Heralding the Next CGM Sensor.

While continuous glucose monitoring (CGM) systems allow precise and real-time blood glucose control, current electrochemicalbased CGM technologies inherently harbor enzyme instability issues. The direct electron transfer (DET) type open circuit potential (OCP) based enzyme sensing principle can minimize the catalytic turnover of the enzyme reaction, thereby providing longer-term operational stability in future CGM glucose sensors. DET-type OCP based glucose sensors were constructed using gold disk electrodes with glucose dehydrogenase capable of DET which was immobilized using a self-assembled monolayer (SAM). The single enzyme layer prepared on the gold electrode was operated in the presence of glucose, using in vitro buffer solution, continuously for over 3 months with the OCP sensor signal monitored every 10 seconds at 25°C. The DET-type OCP glucose sensor was continuously operated for more than 3 months without a significant decrease of the sensor signal and sensitivity (slope). These results suggest that the DET-type OCP glucose sensor is far more stable than the sensor constructed based on the amperometric principle. The long-term stability of DET-type OCP glucose sensor is attributed to the enzyme's minimized catalytic reaction during the operation, thereby extending the lifetime of enzyme. The DET-type OCP glucose sensor can be continuously operated for more than 3 months at 25 °C, in vitro without significant decreases in sensor signal and sensitivity. While the further investigation will be required for in vivo validation, the DET-type OCP glucose sensor is ideal for next generation CGM's, especially in long duration implantable use cases.

Kinetic and Thermodynamic Analysis of Cu2+-Dependent Reductive Inactivation in Direct Electron Transfer-Type Bioelectrocatalysis by Copper Efflux Oxidase

Kinetic and Thermodynamic Analysis of Cu2+-Dependent Reductive Inactivation in Direct Electron Transfer-Type Bioelectrocatalysis by Copper Efflux Oxidase

Electrochemical characterization of a truncated hydrogenase from Pyrococcus furiosus

A highly thermostable cytoplasmic [NiFe] hydrogenase PfSHI, incorporating four subunits (αβγδ), displays a clear direct electron transfer (DET) type bioelectrocatalytic activity. In order to investigate the biological and electrochemical properties of the corresponding truncated hydrogenases, we constructed two truncated enzymes (i.e. Pfαδ and Pfα) derived from the native hydrogenase and immobilized them on multiwalled carbon nanotubes casted glassy carbon electrodes. The cyclic voltammogram along with the enzymatic activity assay revealed that the dimer Pfαδ showed an obvious oxidation current under H2 in a wide temperature range, demonstrating its DET capability, while the monomer Pfα had no activity in H2 oxidation. At high potentials, Pfαδ was proved to be inactive and observed no reverse recovery on the return scan under 40 to 60°C. Notably, Pfαδ still maintained oxygen tolerance to some extent and its activity can be recovered after applying a reduction potential. Further, enzyme turnover frequencies and kinetic parameters were analyzed based on a steady state model, which revealed that Pfαδ exhibited a higher electron transfer rate than that of pristine PfSHI due to the shortened electron transfer distance between the enzyme and the electrode.

A self-powered glucose sensor based on BioCapacitor principle with micro-sized enzyme anode employing direct electron transfer type FADGDH

Diabetes mellitus is a disorder in which the body does not produce enough or respond normally to insulin; consequently, blood glucose levels increase to become abnormally high. Accordingly, the primary treatment of diabetes is to control glycemic levels continuously. To continuously control glycemic levels, several medical devices have been developed to monitor blood glucose levels, represented by sensors and monitors for the self-monitoring of blood glucose. The ultimate goal for those engaged in research to develop medical devices is to develop implantable biodevices, namely self-powered autonomously operated artificial pancreas systems. One of the most challenging issues in realizing an implantable artificial pancreas is the long-term continuous supply of electricity, which is currently dependent on rechargeable batteries, requiring periodical replacement. In this work, we report the development of a direct electron transfer type enzyme-based miniaturized self-powered glucose sensor based on the BioCapacitor principle with a micro-sized enzyme anode area (0.15 mm × 0.75 mm), which has only 0.1 mm2 of electrode surface. As a result, a BioCapacitor utilizing a biofuel cell with a micro-sized enzyme anode was operated by self-power. In addition, the glucose concentration was detected within the range from 13 mM to 100 mM based on the frequency of charge/discharge cycles of the BioCapacitor. Although further improvement of the current density of the micro-sized anode is necessary to monitor a glucose concentration range lower than 13 mM, this self-powered glucose sensor with a micro-sized electrode based on the BioCapacitor principle was operated continuously for 6.6 h at 37 °C in 100 mM potassium phosphate buffer (pH 7.0). Our success indicates the potential to realize self-powered, autonomous, and implantable sensing modules for bio devices such as glucose-sensing systems for an artificial pancreas.

Rational design of direct electron transfer type l-lactate dehydrogenase for the development of multiplexed biosensor

The development of wearable multiplexed biosensors has been focused on systems to measure sweat l-lactate and other metabolites, where the employment of the direct electron transfer (DET) principle is expected. In this paper, a fusion enzyme between an engineered l-lactate oxidase derived from Aerococcus viridans, AvLOx A96L/N212K mutant, which is minimized its oxidase activity and b-type cytochrome protein was constructed to realize multiplexed DET-type lactate and glucose sensors. The sensor with a fusion enzyme showed DET to a gold electrode, with a limited operational range less than 0.5 mM. A mutation was introduced into the fusion enzyme to increase Km value and eliminate its substrate inhibition to construct “b2LOxS”. Together with the employment of an outer membrane, the detection range of the sensor with b2LOxS was expanded up to 10 mM. A simultaneous lactate and glucose monitoring system was constructed using a flexible thin-film multiplexed electrodes with b2LOxS and a DET-type glucose dehydrogenase, and evaluated their performance in the artificial sweat. The sensors achieved simultaneous detection of lactate and glucose without cross-talking error, with the detected linear ranges of 0.5–20 mM for lactate and 0.1–5 mM for glucose, sensitivities of 4.1 nA/mM∙mm2 for lactate and 56 nA/mM∙mm2 for glucose, and limit of detections of 0.41 mM for lactate and 0.057 mM for glucose. The impact of the presence of electrochemical interferants (ascorbic acid, acetaminophen and uric acid), was revealed to be negligible. This is the first report of the DET-type enzyme based lactate and glucose dual sensing systems.

Rational Surface Modification of Carbon Nanomaterials for Improved Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes

Interfacial electron transfer between redox enzymes and electrodes is a key step for enzymatic bioelectrocatalysis in various bioelectrochemical devices. Although the use of carbon nanomaterials enables an increasing number of redox enzymes to carry out bioelectrocatalysis involving direct electron transfer (DET), the role of carbon nanomaterials in interfacial electron transfer remains unclear. Based on the recent progress reported in the literature, in this mini review, the significance of carbon nanomaterials on DET-type bioelectrocatalysis is discussed. Strategies for the oriented immobilization of redox enzymes in rationally modified carbon nanomaterials are also summarized and discussed. Furthermore, techniques to probe redox enzymes in carbon nanomaterials are introduced.

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.

Construction of Super-Stabilized Direct Electron Transfer Type Glucose Dehydrogenase for Long Term Continuous Glucose Sensing System

Introduction: The development of super-stabilized flavin adenine dinucleotide glucose dehydrogenase (FADGDH) was achieved based on the rational biomolecular engineering.The FADGDH from Burkholderia cepacia (BcGDH) is the ideal enzyme for continuous glucose monitoring (CGM) sensor, because of its inherent DET ability (Yamazaki 2008, Lee 2018). BcGDH is composed of three subunits, which are the catalytic subunit harboring FAD and iron-sulfur (3Fe-4S) cluster, the electron transfer subunit harboring three heme c, and the small subunit which functions as a “hitchhiker” protein. We recently reported the BcGDH catalytic subunit complexed with small subunit (Yoshida et al., 2019, PDB 6a2u), thereby elucidated the intra-molecular electron transfer pathway, and inherent disulfide bond between catalytic subunit and small subunit.BcGDH shows two distinct temperature profiles due to variations in its quaternary structure (Sode 1996, Yamazaki 1999). BcGDH composed of three subunits showed an optimum reaction temperature of approximately 45 ̊C. By increasing temperature, enzyme activity gradually decreases, due to the dissociation of electron transfer subunit. However, at temperature above 60 ̊C, enzyme activity increases again with the increase the temperature and shows second peak at around 70 ̊C, which is derived from the complex composed of catalytic subunit and small subunit. This phenomenon suggested that the dissociation of the electron transfer subunit dominates the stability of BcGDH three subunit complex, which is the essential structure to show DET ability. Namely, to stabilize BcGDH enzyme complex, the stabilization of quaternary structure is the most rational strategy for this enzyme.We recently succeeded in the elucidation of crystal structure of BcGDH complex together with the electron transfer subunit. The availability of quaternary structure of this enzyme enabled us to design inter-subunit disulfide bond in BcGDH. In this study, the designing and characterization of super-stabilized BcGDH is presented. We also characterize the DET ability of the engineered BcGDH, which is the mandatory characteristic of this enzyme for the development of the future innovative biosensing systems. Methods: Amino acid substitution to cysteine residues were carried out at the residues existing on the surface of each subunit, to form inter-subunit disulfide bonds. Recombinantly prepared BcGDH mutant complexes were purified by affinity chromatography. The catalytic activity and thermal stability were investigated. Then DET-type sensors were constructed by immobilization on the gold electrode with self-assembled monolayer (SAM) layer, and investigated their stability by continuous operation. Results and Discussion: Based on the 3D structure of BcGDH complex, disulfide bonds between electron transfer subunit and either catalytic subunit or small subunit were designed. By the screening of the cystaine residue substituted mutants, some mutants showed remarkable high thermal stability as BcGDH complex without the decrease of the catalytic activity. Furthermore, multiple inter-subunit disulfide bonds made the enzyme as super-stabilized which showed high catalytic activity even by the incubation at 70 ̊C. Thus developed engineered enzyme revealed to show DET ability with electrode, indistinguishable to the wild type one. This super-stabilized enzyme will contribute the improvement of the operational stability of enzyme sensors. References Lee et al., Bioelectrochemistry. 121 (2018) 1–6Sode et al., Enzyme Microb. Technol. 19 (1996) 82–85Yamazaki et al., Appl. Biochem. Biotechnol. 77 (1999) 325–336Yamazaki et al., Anal. Lett. 41 (2008) 2363-2373.Yoshida et al., Acta Crystallogr. Sect. D Struct. Biol. 75 (2019) 841–851

An amperometric biosensor of L-fucose in urine for the first screening test of cancer

Quantitative routine detection of fucose, which is a cancer marker, in urine is effective for the preliminary screening of cancer. Amperometric biosensing methods have the advantage of being simple, rapid, and precise for urinalysis. However, coexisting electroactive interferences such as ascorbic acid (AA), dopamine (DA), and uric acid (UA) prevent accurate measurements. In this work, an amperometric l-fucose biosensor unaffected by interferences was developed and utilizes direct electron transfer type bioelectrocatalysis of pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH). The isolated PQQ domain from CcPDH was immobilized on gold nanoparticle (AuNP)-modified electrodes, which obtained a catalytic current at a lower potential than the oxidation potential of the interfering compounds. Applying an operating potential of −0.1 V vs. Ag|AgCl (3 M NaCl) enabled the detection of l-fucose while completely eliminating the oxidation of AA, DA, and UA on the electrodes. The increase in the specific area of the electrodes by increasing the AuNP drop-casting time resulted in an improvement in the sensor performance. The biosensor exhibited a linear range for l-fucose detection between 0.1 mM and 1 mM (R2 = 0.9996), including a cut-off value, the sensitivity was 3.12 ± 0.05 μA mM−1 cm−2, and the detection limit was 13.6 μM at a signal-to-noise ratio of three. The biosensor can be used to quantify the concentration of l-fucose at physiological levels and does not require urine preprocessing, making it applicable to practical use for point-of-care testing with urine.

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

(Invited) Mediator-Free Electron-Transfer on Flexible and Patternable Biointerface for Sweat Glucose and Surface Electromyography Monitoring

Introduction Wearable electrochemical biosensors are widely applied to detect biochemical molecules related to physiology and metabolism in biofluids such as sweat, tear and saliva.[1] These electrochemical biosensors are mainly based on enzyme-electrochemical reactions with high specificity at enzyme-electrode interfaces to transduce the molecular signal to an electrical signal. [2] Therefore, electron transfer (ET) at the enzyme-electrode interface is the key step for such wearable electrochemical biosensors.[3] Most current reported wearable electrochemical biosensors often employed extra electron mediators to enhance the enzyme-electrochemical reactions and to achieve efficient ET.[4] However, the use of electron mediators not only increases the complexity of biosensors but also leads to low selectivity and instability due to the cross-reaction and the leakages of electron mediators, respectively.[5] Therefore, the realization of direct ET at enzyme-electrode interfaces without using electron mediators to develop a wearable electrochemical biosensor with high selectivity and sensitivity for biochemical molecules monitoring is highly appreciated. Method Conductive CNT-EVA film was prepared by well dispersing CNT in xylene and then mixed through being sonicated to form a homogeneous ink. Then CNT-EVA ink was casted into a glass substrate and dried at room temperature to remove the solvent totally, to prepare flexible CNT-EVA films. The enzymatic bioelectrodes were prepared by dropping 50 μL aliquot of an enzyme/reagent mixture containing HRP (usually 0.25 mg mL-1), GOx (2.5 mg mL-1), and glutaraldehyde (2.5%) onto the flexible CNT-EVA film surface and dried at 4 °C for 2 h.All electrochemical measurements were carried out by an electrochemical analyzer CHI660D (Shanghai Chenhua, China), a three-electrode system constituted with a CNT-EVA work electrode, an Ag|AgCl|sat. KCl reference electrode and a Pt wire counter electrode was utilized. Surface electromyography (sEMG) signal was captured by a homemade eight-channel EMG amplifier with a gain of 4000, and then sampled by a data converter (DAQ-6341, National Instruments, Austin, TX, USA) at a sample rate of 1000 Hz with a 16-bit resolution. Results and Conclusions A flexible, conductive and hierarchical meso/macro porous carbon nanotubes (CNT) - ethylene-vinyl acetate copolymer (EVA) film is constructed as the sensing substrate in a facile route. Thanks to its well-formed three-dimensional nanoporous structure, direct electron transfer-type of bioelectrocatalysis is successfully realized on the glucose oxidase-horseradish peroxidase bienzyme functionalized CNT-EVA film for glucose monitoring without any mediator. The as-fabricated biosensors show excellent selectivity to glucose with a super-high sensitivity of 270 ± 10 μA mM-1 cm-2. To the best of the authors' knowledge, this might be the highest sensitivity of the flexible enzymatic biosensor for glucose monitoring demonstrated so far. This flexible CNT-EVA films also are utilized for monitoring the sEMG with high signal-to-background sensitivity due to its high electrical conductivity as well as good conformability to the skin. On-body experiments illustrated the capability of the proposed flexible biosensor for simultaneously monitoring sweat glucose and sEMG, guiding the way for measuring multiplex physiological signals for health monitoring, early diagnosis, and clinical research in future.

Mediator-free electron-transfer on patternable hierarchical meso/macro porous bienzyme interface for highly-sensitive sweat glucose and surface electromyography monitoring

Wearable electrochemical biosensors, which measure the concentration of small biochemical molecules in biofluids in a non-invasive way, are still highly desired. Herein, a wearable, mediator-free electrochemical biosensor for real-time sweat glucose monitoring is proposed. A flexible and hierarchical meso/macro porous carbon nanotubes (CNT) - ethylene-vinyl acetate copolymer (EVA) film is constructed as the sensing substrate in a facile route. Thanks to its well-formed three-dimensional conductive nanoporous structure, direct electron transfer-type of bioelectrocatalysis is successfully realized on the glucose oxidase-horseradish peroxidase bienzyme functionalized CNT-EVA film for glucose monitoring without any mediator. The as-fabricated biosensors show excellent selectivity to glucose with a super-high sensitivity of 270 ± 10 μA mM−1 cm−2. This flexible CNT-EVA films also are utilized for monitoring the surface electromyography (sEMG) with high signal-to-background sensitivity due to its high electrical conductivity as well as good conformability to the skin. On-body experiments illustrated the capability of the proposed flexible biosensor for monitoring sweat glucose and surface electromyography simultaneously, guiding the way for measuring multiplex physiological signals for health monitoring, early diagnosis, and clinical research in future.