Insight into continuous glucose monitoring: from medical basics to commercialized devices.

According to the latest statistics, more than 537 million people around the world struggle with diabetes and its adverse consequences. As well as acute risks of hypo- or hyper- glycemia, long-term vascular complications may occur, including coronary heart disease or stroke, as well as diabetic nephropathy leading to end-stage disease, neuropathy or retinopathy. Therefore, there is an urgent need to improve diabetes management to reduce the risk of complications but also to improve patient's quality life. The impact of continuous glucose monitoring (CGM) is well recognized, in this regard. The current review aims at introducing the basic principles of glucose sensing, including electrochemical and optical detection, summarizing CGM technology, its requirements, advantages, and disadvantages. The role of CGM systems in the clinical diagnostics/personal testing, difficulties in their utilization, and recommendations are also discussed. In the end, challenges and prospects in future CGM systems are discussed and non-invasive, wearable glucose biosensors are introduced. Though the scope of this review is CGMs and provides information about medical issues and analytical principles, consideration of broader use will be critical in future if the right systems are to be selected for effective diabetes management.

From fundamentals to applications of bioelectrocatalysis: bioelectrocatalytic reactions of FAD-dependent glucose dehydrogenase and bilirubin oxidase.

In this review, I present the main highlights of my works in the development of bioelectrocatalysis, which can be used in widespread applications, particularly for the design of biosensor and biofuel cells. In particular, I focus on research progress made in two key bioelectrocatalytic reactions: glucose oxidation by flavin adenine dinucleotide-dependent glucose dehydrogenase and oxygen reduction by bilirubin oxidase. I demonstrate the fundamental principles of bioelectrocatalysis and the requirements for enhancing the catalytic performance, including the choice of a mediator of redox reactions, immobilization, and electrode materials. These methods can allow for achieving control of the bioelectrocatalytic reaction, thereby overcoming obstacles toward their industrial applications.

Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors.

The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus species require suitable redox mediators to transfer electrons from the enzyme to the electrode surface for the application of bioelectrical devices. Although several mediators for FAD-GDH are already in use, they are still far from optimum in view of potential, kinetics, sustainability, and cost-effectiveness. Herein, we investigated the efficiency of various phenothiazines and quinones in the electrochemical oxidation of FAD-GDH from Aspergillus terreus. At pH 7.0, the logarithm of the bimolecular oxidation rate constants appeared to depend on the redox potentials of all the mediators tested. Notably, the rate constant of each molecule for FAD-GDH was approximately 2.5 orders of magnitude higher than that for glucose oxidase from Aspergillus sp. The results suggest that the electron transfer kinetics is mainly determined by the formal potential of the mediator, the driving force of electron transfer, and the electron transfer distance between the redox active site of the mediator and the FAD, affected by the steric or chemical interactions. Higher k2 values were found for ortho-quinones than for para-quinones in the reactions with FAD-GDH and glucose oxidase, which was likely due to less steric hindrance in the active site in the case of the ortho-quinones.

Exceptionally high glucose current on a hierarchically structured porous carbon electrode with "wired" flavin adenine dinucleotide-dependent glucose dehydrogenase.

This article introduces a carbon electrode designed to achieve efficient enzymatic electrolysis by exploiting a hierarchical pore structure based on macropores for efficient mass transfer and mesopores for high enzyme loading. Magnesium oxide-templated mesoporous carbon (MgOC, mean pore diameter 38 nm) was used to increase the effective specific surface area for enzyme immobilization. MgOC particles were deposited on a current collector by an electrophoretic deposition method to generate micrometer-scale macropores to improve the mass transfer of glucose and electrolyte (buffer) ions. To create a glucose bioanode, the porous-carbon-modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, deglycosylated flavin adenine dinucleotide-dependent glucose dehydrogenase (d-FAD-GDH), and a cross-linker. Carbohydrate chains on the peripheral surfaces of the FAD-GDH molecules were removed by periodate oxidation before cross-linking. The current density for the oxidation of glucose was 100 mA cm(-2) at 25 °C and pH 7, with a hydrogel loading of 1.0 mg cm(-2). For the same hydrogel composition and loading, the current density on the MgOC-modified electrode was more than 30 times higher than that on a flat carbon electrode. On increasing the solution temperature to 45 °C, the catalytic current increased to 300 mA cm(-2), with a hydrogel loading of 1.6 mg cm(-2). Furthermore, the stability of the hydrogel electrode was improved by using the mesoporous carbon materials; more than 95% of the initial catalytic current remained after a 220-day storage test in 4 °C phosphate buffer, and 80% was observed after 7 days of continuous operation at 25 °C.

Lactate biosensors: current status and outlook.

Many research efforts over the last few decades have been devoted to sensing lactate as an important analytical target in clinical care, sport medicine, and food processing. Therefore, research in designing lactate sensors is no longer in its infancy and now is more directed toward viable sensors for direct applications. In this review, we provide an overview of the most immediate and relevant developments toward this end, and we discuss and assess common transduction approaches. Further, we critically describe the pros and cons of current commercial lactate sensors and envision how future sensing design may benefit from emerging new technologies.

Nature-Inspired Superhydrophilic Biosponge as Structural Beneficial Platform for Sweating Analysis Patch.

Perspiration plays a pivotal role not only in thermoregulation but also in reflecting the body's internal state and its response to external stimuli. The up-to-date skin-based wearable platforms have facilitated the monitoring and simultaneous analysis of sweat, offering valuable physiological insights. Unlike conventional passive sweating, dynamic normal perspiration, which occurs during various activities and rest periods, necessitates a more reliable method of collection to accurately capture its real-time fluctuations. An innovative microfluidic patch incorporating a hierarchical superhydrophilic biosponge, poise to significantly improve the efficiency capture of dynamic sweat is introduced. The seamlessly integrated biosponge microchannel showcases exceptional absorption capabilities, efficiently capturing non-sensitive sweat exuding from the skin surface, mitigating sample loss and minimizing sweat volatilization. Furthermore, the incorporation of sweat-rate sensors alongside a suite of functional electrochemical sensors endows the patch of uninterrupted monitoring and analysis of dynamic sweat during various activities, stress events, high-energy intake, and other scenarios.

Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center.

The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH···S(-)(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)···O(-)(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gln. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper.

Direct electron transfer to a metagenome-derived laccase fused to affinity tags near the electroactive copper site.

We demonstrate the efficient direct electron transfer (DET) from an electrode to an engineered laccase isolated from a metagenome. The enzyme has a unique homotrimeric architecture with a two-domain-type laccase subunit. The recombinant laccase-modified mesoporous carbon electrode exhibits an effective catalytic current for oxygen reduction, which depends on the affinity tags attached near the electroactive Cu site of the enzyme. We also investigated the effect of the affinity tags on the orientation of the enzyme on functional thiol-modified Au electrodes. The results suggest that a poly-histidine tag (His-tag) functions as an anchor to control the orientation of the enzyme to enhance the current density of the DET-type bioelectrocatalysis.

Bioelectrocatalytic oxidation of glucose with antibiotic channel-containing liposomes.

We developed an efficient bioelectrocatalytic system for glucose oxidation by introducing hydrophilic glucose-permeable antibiotic channels into liposomes.

Hierarchical Structure of Gold and Carbon Electrode for Bilirubin Oxidase-Biocathode.

Biofuel cells (BFCs) with enzymatic electrocatalysts have attracted significant attention, especially as power sources for wearable and implantable devices; however, the applications of BFCs are limited owing to the limited O2 supply. This can be addressed by using air-diffusion-type bilirubin oxidase (BOD) cathodes, and thus the further development of the hierarchical structure of porous electrodes with highly effective specific surface areas is critical. In this study, a porous layer of gold is deposited over magnesium-oxide-templated carbon (MgOC) to form BOD-based biocathodes for the oxygen reduction reaction (ORR). Porous gold structures are constructed via electrochemical deposition of gold via dynamic hydrogen bubble templating (DHBT). Hydrogen bubbles used as a template and controlled by the Coulomb number yield a porous gold structure during the electrochemical deposition process. The current density of the ORR catalyzed by BOD without a redox mediator on the gold-modified MgOC electrode was 1.3 times higher than that of the ORR on the MgOC electrode. Furthermore, the gold-deposited electrodes were modified with aromatic thiols containing negatively charged functional groups to improve the orientation of BOD on the electrode surface to facilitate efficient electron transfer at the heterogeneous surface, thereby achieving an ORR current of 12 mA cm-2 at pH 5 and 25 °C. These results suggest that DHBT is an efficient method for the fabrication of nanostructured electrodes that promote direct electron transfer with oxidoreductase enzymes.

A disposable enzymatic biofuel cell for glucose sensing via short-circuit current.

It is essential to construct a biofuel cell-based sensor and develop an effective strategy to detect glucose without any potentiostat circuitry in order to create a simple and miniaturized device. In this report, an enzymatic biofuel cell (EBFC) is fabricated by the facile design of an anode and cathode on a screen-printed carbon electrode (SPCE). To construct the anode, thionine and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) are covalently immobilized via a crosslinker to make a cross-linked redox network. As a cathode, the Pt-free oxygen reduction carbon catalyst is employed alternative to the commonly used bilirubin oxidase. We proposed the importance of EBFC-based sensors through the connection of anode and cathode; they can identify a short-circuit current by means of applied zero external voltage, thereby capable of glucose detection without under the operation of the potentiostat. The result shows that the EBFC-based sensor could be able to detect based on a short-circuit current with a wide range of glucose concentrations from 0.28 to 30 mM. Further, an EBFC is employed as a one-compartment model energy harvester with a maximum power density of (36 ± 3) µW cm- 2 in sample volume 5 µL. In addition, the constructed EBFC-based sensor demonstrates that the physiological range of ascorbic acid and uric acid shows no significant effect on the short-circuit current generation. Moreover, this EBFC can be used as a sensor in artificial plasma without losing its performance and thereby used as a disposable test strip in real blood sample analysis.

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells.

Nanomaterials, including carbon nanotubes, graphene oxide, metal-organic frameworks, metal nanoparticles, and porous carbon, play a crucial role as efficient carriers to enhance enzyme activity through substrate channeling while improving enzyme stability and reusability. However, there are significant debates surrounding aspects such as enzyme orientation, enzyme loading, retention of enzyme activity, and immobilization techniques. Consequently, these subjects have become the focus of intensive research in the realm of multi-enzyme cascade reactions. Researchers have undertaken the challenge of creating functional in vitro multi-enzyme systems, drawing inspiration from natural multi-enzyme processes within living organisms. Substantial progress has been achieved in designing multi-step reactions that harness the synthetic capabilities of various enzymes, particularly in applications such as biomarker detection (e.g., biosensors) and the development of biofuel cells. This review provides an overview of recent developments in concurrent and sequential approaches involving two or more enzymes in sequence. It delves into the intricacies of multi-enzyme cascade reactions conducted on nanostructured electrodes, addressing both the challenges encountered and the innovative solutions devised in this field.

High-performance paper-based biocathode fabricated by screen-printing an improved mesoporous carbon ink and by oriented immobilization of bilirubin oxidase.

In this study, the performance of a paper-based, screen-printed biofuel cell with mesoporous MgO-templated carbon (MgOC) electrodes was improved in two steps. First, a small amount of carboxymethyl cellulose (CMC) was added to the MgOC ink. Next, the cathode was modified with bilirubin prior to immobilizing the bilirubin oxidase (BOD). The CMC increased the accessibility of the mesopores of the MgOC, and subsequently, the performance of both the bioanode and biocathode. CMC also likely increased the stability of the electrodes. The pre-modification with bilirubin improved the orientation of the BOD, which facilitated direct electron transfer. With these two steps, an open circuit potential of 0.65 V, a maximal current density of 1.94 mA cm-2, and a maximal power density of 465 µW cm-2 was achieved with lactate oxidase as bioanode enzyme and lactate as fuel. This is one of the highest reported performances for a biofuel cell.

Efficient direct electron transfer of PQQ-glucose dehydrogenase on carbon cryogel electrodes at neutral pH.

We present a comprehensive study of the direct electron transfer reaction of soluble PQQ-GDH from Acinetobacter calcoaceticus. Wild-type PQQ-sGDH nonspecifically adsorbed on carbon cryogel electrodes retained its enzymatic activity for glucose and maltose oxidation at pH 7.2 and 37 °C. The cyclic voltammograms in the absence of enzymatic substrate showed 2 redox peaks that suggest a two-step, one-electron oxidation/reduction of PQQ. Calibration curves showed a linear amperometric response for a wide glucose concentration range, including the values normally found in blood. At saturation, the catalytic current reached 0.93 mA cm(-2). Altogether the experimental results suggest that the amperometric output of the electrodes and the shape of the calibration curves represent a combination of the intrinsic enzyme kinetics, the maximum rate of heterogeneous electron transfer and the substrate accessibility to the enzyme's active center caused by the confinement of the enzyme into the mesoporous structure. A new mutant enzyme, N428C, developed in our group that shows almost twice the maximum catalytic activity in homogeneous experiments in solution, also showed a DET signal on carbon cryogel electrodes for glucose electro-oxidation. The higher activity for the mutant enzyme was also verified on the electrode surface.

Designing a cross-linked redox network for a mediated enzyme-based electrode.

A bio-conjugated redox network matrix based on glucose dehydrogenase, thionine (diamine-containing mediator), and poly(ethylene glycol) diglycidyl ether (crosslinker) is developed on a glassy carbon electrode through covalent bonding with one-pot crosslinking. Electrons from the enzyme diffuse through the network producing 400 µA cm-2 of glucose oxidation current at 25 °C.

Disposable electrochemical glucose sensor based on water-soluble quinone-based mediators with flavin adenine dinucleotide-dependent glucose dehydrogenase.

Glucose level measurement is essential for the point-of-care diagnosis, primarily for persons with diabetes. A disposable electrochemical glucose sensor is constructed using flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and redox mediator for electron transfer from the enzyme to the electrode surface. Ideally, a suitable mediator should have high water solubility, high kinetic constant, high stability, and redox potential between -0.2 and 0.1 V vs. Ag|AgCl|sat. KCl. We designed and synthesized two new quinone-based water-soluble mediators: quinoline-5,8-dione (QD) and isoquinoline-5,8-dione (IQD). The formal potentials for both QD and IQD at pH 7.0 were -0.07 V vs. Ag|AgCl|sat. KCl. The logarithms of the electron exchange rate constants (k2/(M-1 s-1)) between QD/IQD and FAD-GDH were 7.7 ± 0.1 and 7.4 ± 0.1 for QD and IQD, respectively, which are the highest value among the water-soluble mediators for FAD-GDH reported to date. Disposable amperometric glucose sensors were fabricated by dropping FAD-GDH and QD or IQD onto a test strip. The sensor achieved a linear response up to glucose concentrations of 55.5 mM. The linear response was obtained even when the mediator loading was low (0.5 nmol/strip); loading was only 0.2 mol% of glucose. The results proved that the response current was primarily controlled by glucose diffusion. In addition, the sensor using QD exhibited high stability over 3 months at room temperature.

Extracellular electron transfer by Microcystis aeruginosa is solely driven by high pH.

Extracellular electron transfer (EET) by the cyanobacterium Microcystis aeruginosa was investigated. Observations indicate that EET onto an electrode poised at + 0.6 vs. standard hydrogen electrode (SHE) is triggered by high pH, more evidently at pH levels above 9. Light intensity does not appear to affect electricity generation, indicating that this may not be a "biophotovoltaic" process. The generated current density was amplified with stepwise pH increases from approximately 5 mA m-2 at pH 7.8 to 30 mA m-2 at pH 10.5, for dense (0.4 mg mL-1 dry weight) Microcystis aeruginosa suspensions with dissolved CO2 and O2 approaching equilibrium with atmospheric concentrations. The upsurge in current density was more pronounced (from 5 mA m-2 at pH 7.8 to 40 mA m-2 at pH 10.2) in the absence of the cells' natural electron acceptors, dissolved CO2 and O2. However, the latter effect is more likely due to competition for electrons by oxygen than to reductive stress. EET in this species is therefore a light-independent process that is enhanced by increasing pH, with reasons that are still unknown, but either related to the involvement of protons in the last step of electron transfer, or to intracellular pH control.

Self-Powered Diaper Sensor with Wireless Transmitter Powered by Paper-Based Biofuel Cell with Urine Glucose as Fuel.

A self-driven sensor that can detect urine and urine sugar and can be mounted on diapers is desirable to reduce the burden of long-term care. In this study, we created a paper-based glucose biofuel cell that can be mounted on diapers to detect urine sugar. Electrodes for biofuel cells were produced by printing MgO-templated porous carbon on which poly(glycidyl methacrylate) was modified using graft polymerization. A new bioanode was prepared through covalently modifying flavin-adenine-dinucleotide-dependent glucose dehydrogenase and azure A with pendant glycidyl groups of poly(glycidyl methacrylate). We prepared a cathode with covalently bonded bilirubin oxidase. Covalent bonding of enzymes and mediators to both the bioanode and biocathode suppressed elution and improved stability. The biofuel cell could achieve a maximum output density of 0.12 mW cm-2, and by combining it with a wireless transmission device, the concentration of glucose sensed from the transmission frequency was in the range of 0-10 mM. The sensitivity of the sensor was estimated at 0.0030 ± 0.0002 Hz mmol-1 dm3. This device is expected to be a new urine-sugar detection device, composed only of organic materials with a low environmental load and it can be useful for detecting postprandial hyperglycemia.

Chitosan-based enzyme ink for screen-printed bioanodes.

In this study, magnesium oxide (MgO)-templated mesoporous carbon (MgOC) and chitosan cross-linked with genipin (chitosan-genipin) were considered bio-composite inks for screen-printed bioanodes. The fabrication processes were optimized using rheological and structural data, and a bioanode ink containing glucose oxidase (GOx) and 1,2-naphthoquinone (1,2-NQ) was successfully developed. The optimal bioanode-ink contained MgOC pre-treated by washing to achieve a hydrophilic and neutral surface, which helped maintain enzyme activity and resulted in a highly porous electrode structure, which is essential for the accessibility of glucose to GOx. A bioanode fabricated using this ink showed a linear response current dependency up to 8 mM glucose with a sensitivity of 25.83 µA cm-2 mM-1. Combined with a conventional biocathode, an electromotive force of 0.54 V and a maximal power density of 96 µW cm-2 were achieved. These results show that this bio-composite ink can be used to replace the multi-step process of printing with conventional ink followed by drop-casting enzyme and mediator with a one-step printing process.

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 A resolution using a twinned crystal.

Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6(3). Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33-2.3 A. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD.