Flavin Adenine Dinucleotide Glucose Dehydrogenase Research Articles

Low-Potential Operation of Direct Electron Transfer Biosensor Strip With Single-Walled Carbon Nanotubes and Flavin Adenine Dinucleotide Glucose Dehydrogenase

In this study, the low-potential operation (+0.05 V vs. Ag/AgCl) of a direct electron transfer (DET) glucose biosensor strip with a small-volume (2 μL) electrochemical capillary chamber is demonstrated. We constructed a DET formation in which de-bundled single-walled carbon nanotubes (SWCNTs) were placed near the reaction center pocket of flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH). The DET biosensor has the advantage of long-term stability, unlike an electron transfer mediator, which is naturally reduced to cause sensing malfunction in a mediated electron transfer biosensor, adopted in commercial devices. However, DET biosensors still have a high working potential owing to their overpotential. In this article, we have resolved this issue via a very short interval (1 ms) voltage application (+0.4 V) that reduced the onset potential, in other words, enhanced the current owing to an enzymatic reaction. The DET biosensors had a sensitivity of 0.055 μA (mg/dl) <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-2</sup> , a linear dynamic range of 0-750 mg/dL, and a detection limit of 0.9 mg/dL.

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.

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.

Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors

A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).

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

Xylose-Insensitive Direct Electron Transfer Biosensor Strip With Single-Walled Carbon Nanotubes and Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase

Commercial biosensor strip for self-blood sugar monitoring for diabetes is type of mediated electron transfer (MET), where an electron transfer mediator is used. Here, a xylose-insensitive direct electron transfer (DET) glucose biosensor strip is developed for the first time. It overcomes the disadvantages of MET counterparts. A DET formation in which single-walled carbon nanotubes (SWCNTs), debundled using sodium cholate (SC) surfactant, are positioned near the reaction center pocket of flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH) is constructed. Concomitantly, it is found that thymol (TM) increases the DET current seven times compared to when it is not used. TM does not have a specific redox potential and does not work without carbon nanotubes (CNTs), but assisted DET between the CNTs and FAD-GDH. Therefore, it is not an electron transfer mediator but a DET enhancement chemical. Long-term storage tests show that GDH/TM/SWCNT-SC electrodes are not naturally reduced unlike MET biosensor electrodes. The “bionanoink,” composed of glycan chain rich novel fungal FAD-GDH, SWCNTs, SC, and TM in aqueous solution is suitable for the fabrication of a DET biosensor strip that is insensitive to xylose. The resulting DET biosensor strip had the required sample volume of 900 nL (1.5 × 0.1 × 6.0 mm), a measurement range of 0-600 mg/mL, measurement time of 10 s, the ability for repeated measurements, and a 5% coefficient of variation in real sample measurements. A low cost biosensor will be widely spread, consequently, will contribute to the realization of a healthy society.

X-ray structure of the direct electron transfer-type FAD glucose dehydrogenase catalytic subunit complexed with a hitchhiker protein.

The bacterial flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe-4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound β-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.

Minimally Invasive Glucose Monitoring Using a Highly Porous Gold Microneedles-Based Biosensor: Characterization and Application in Artificial Interstitial Fluid

In this paper, we present the first highly porous gold (h-PG) microneedles-based second-generation biosensor for minimally invasive monitoring of glucose in artificial interstitial fluid (ISF). A highly porous microneedles-based electrode was prepared by a simple electrochemical self-templating method that involves two steps, gold electrodeposition and hydrogen bubbling at the electrode, which were realized by applying a potential of −2 V versus a saturated calomel electrode (SCE). The highly porous gold surface of the microneedles was modified by immobilization of 6-(ferrocenyl)hexanethiol (FcSH) as a redox mediator and subsequently by immobilization of a flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH) enzyme using a drop-casting method. The microneedles-based FcSH/FAD-GDH biosensor allows for the detection of glucose in artificial interstitial fluid with an extended linear range (0.1–10 mM), high sensitivity (50.86 µA cm−2 mM−1), stability (20% signal loss after 30 days), selectivity (only ascorbic acid showed a response about 10% of glucose signal), and a short response time (3 s). These properties were favourably compared to other microneedles-based glucose biosensors reported in the literature. Finally, the microneedle-arrays-based second-generation biosensor for glucose detection was tested in artificial interstitial fluid opportunely spiked with different concentrations of glucose (simulating healthy physiological conditions while fasting and after lunch) and by placing the electrode into a simulated chitosan/agarose hydrogel skin model embedded in the artificial ISF (continuous glucose monitoring). The obtained current signals had a lag-time of about 2 min compared to the experiments in solution, but they fit perfectly into the linearity range of the biosensor (0.1–10 mM). These promising results show that the proposed h-PG microneedles-based sensor could be used as a wearable, disposable, user-friendly, and automated diagnostic tool for diabetes patients.

Diameter dependence of single-walled carbon nanotubes with flavin adenine dinucleotide glucose dehydrogenase for direct electron transfer bioanodes

The diameter dependence of single-walled carbon nanotubes (SWCNTs) with flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH) for direct electron transfer (DET) type bioanodes is presented for the first time. In order to address the issue, we use SWCNTs having different diameters fabricated by direct-injection pyrolytic synthesis, which can control the diameter in a wide range with the other parameters fixed. Three different diameters of SWCNTs are examined: average diameter dm = 1.0, 1.5, and 2.0 nm. In order to achieve DET, a debundled, individual SWCNT has to be placed at the FAD position in the GDH protein pocket. The bioanodes with dm = 1.0 and 1.5 nm SWCNTs show better performance than that with dm = 2.0 nm SWCNTs. The diameter dependence of SWCNTs for bioanode performance is thought to be related to the distance between the SWCNTs and FAD positioned in the reaction center of the GDH protein shell.

Effects of electrolyte on the mediated electrocatalytic glucose oxidation reaction catalyzed by flavin adenine dinucleotide glucose dehydrogenase

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) become a key enzyme for electrochemical oxidation of glucose. Here, we investigated the effects of electrolyte, especially at high concentration, on the bioelectrocatalytic activity of FAD-GDH from Aspergillus terreus using a ferricyanide as a redox mediator. In the low ionic strength region, an increase of bimolecular reaction rate constant (k2) between FAD-GDH and ferricyanide was observed due to the reduction of the ionic repulsive interaction between negatively charged enzyme and mediator. Above 0.5 M concentration, ion-specific increments in k2 were observed; ammonium ions most efficiently increased k2, owing to adsorption via dispersion force on the enzyme surface likely proximal to the active site.

Effect of individual plasma components on the performance of a glucose enzyme electrode based on redox polymer mediation of a flavin adenine dinucleotide-dependent glucose dehydrogenase

The performance of glucose enzyme electrodes, consisting of crosslinked flavin adenine dinucleotide glucose dehydrogenase (FADGDH), an osmium redox polymer and multi-walled carbon nanotubes on graphite electrodes, was tested in phosphate buffered saline, artificial plasma and the individual components of artificial plasma to assess the effect of each component on current response and operational stability of the response to better understand the decrease in electrode performance observed in blood. Electrodes tested in artificial plasma show a significant decrease in current response in 5 mM glucose, and operational stability of the response in 100 mM glucose, compared to electrodes tested in buffer. The lowest current response for the enzyme electrodes was observed in the presence of physiological level of uric acid although the largest alteration to enzyme affinity, as estimated from the apparent Michaelis-Menten constant, occurred upon addition of physiological level of sodium bicarbonate. The operational stability observed in the presence of uric acid was the lowest of all components tested, with only 46% of initial current response after 12 h, and was comparable to the 27% of current remaining after 12 h for electrodes operating in artificial plasma. The effect of uric acid on glucose oxidation by enzyme electrodes prepared using both glucose oxidase (GOx) and a recombinant cellobiose dehydrogenase (CDH) was assessed. The maximum current decreased for both FADGDH and GOx enzyme electrodes in the presence of uric acid, with no significant change to the enzyme affinity, suggesting non-competitive inhibition. The CDH based electrodes provided highest stability of current signal in buffer, with 86% of the initial signal present after 12 h, but display significant change in enzyme affinity, maximum current and operational stability, dropping to only 33%, in the presence of uric acid. In contrast the operational stability of the GOx-based enzyme electrodes was unaffected by the presence of physiological level of uric acid. As uric acid and sodium bicarbonate are present in blood, these results highlight the importance of enzyme selection for in vivo biosensing and biofuel cell applications. Further work is required to understand the mechanism of uric acid inhibition on each of the enzymes.

Construction and characterization of flavin adenine dinucleotide glucose dehydrogenase complex harboring a truncated electron transfer subunit

One of the most prominent glucose dehydrogenases (GDHs) capable of direct electron transfer with electrodes is the FADGDH complex derived from Burkholderia cepacia. This FADGDH complex comprises the following three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, a hitch-hiker protein from the bacterial TAT secretion system (γ subunit), and the electron transfer subunit (β subunit). The electron transfer subunit (β subunit) of the FADGDH complex is a three-heme c containing cytochrome c like molecule (heme 1, heme 2 and heme 3 from the N-terminal). In this study, an FADGDH complex harboring a truncated electron transfer subunit composed of only heme 3 was constructed, and its enzymatic activity and electrochemical properties were investigated to elucidate the role of heme 3 and its region. A truncated electron transfer subunit, trβ subunit, was designed using the 3D structures of homologous cytochrome c proteins. The designed trβ subunit was expressed as soluble and functional heme c molecules forming complexes with γα catalytic complexes. Thus, the formed FADGDH complex has inter-molecular electron transfers from the FAD to the trβ subunit, and from the trβ subunit to the external electron acceptor, showing electron transfer subunit-mediated characteristic dye-mediated dehydrogenase activity with a Ru-complex. Therefore, heme 3 in the electron transfer subunit is responsible for accepting the electron from the γα catalytic complex. Moreover, the FADGDH complex harboring the trβ subunit showed DET activity toward the electrode. Spectroelectrochemical observations revealed that the trβ subunit possessed a lower formal potential than any of the 3 hemes in the whole electron transfer subunit. These unexpected electrochemical properties of the heme in the trβ subunit may potentially result in the construction of a DET principle-based glucose sensor, which can be operated at a much lower potential than those achieved using the FADGDH complex with a whole electron transfer subunit.

Design of Experiments Approach to Provide Enhanced Glucose‐oxidising Enzyme Electrode for Membrane‐less Enzymatic Fuel Cells Operating in Human Physiological Fluids

AbstractGraphite electrodes are modified with a redox polymer, [Os(4,4′‐dimethoxy‐2,2′‐bipyridine)2(polyvinylimidazole)10Cl]+ (E°′=−0.02 V vs Ag/AgCl (3 M KCl), crosslinked with a flavin adenine dinucleotide glucose dehydrogenase and multi‐walled carbon nanotubes for electrocatalytic oxidation of glucose. The enzyme electrodes provide 52 % higher current density, 1.22±0.10 mA cm−2 in 50 mM phosphate‐buffered saline at 37 °C containing 5 mM glucose, when component amounts are optimised using a design of experiments approach compared to one‐factor‐at‐a‐time. Current densities of 0.84±0.15 mA cm−2 were achieved in the presence of oxygen for these enzyme electrodes. Further analysis of the model allowed for altering of the electrode components while maintaining similar current densities, 0.78±0.11 mA cm−2 with 34 % less enzyme. Application of the cost‐effective anodes in membrane‐less enzymatic fuel cells is demonstrated by connection to cathodes prepared by co‐immobilisation of [Os(2,2′‐bipyridine)2(polyvinylimidazole)10Cl+] redox polymer, Myrothecium verrucaria bilirubin oxidase and multi‐walled carbon nanotubes on graphite electrodes. Power densities of up to 285 μW cm−2, 146 μW cm−2 and 60 μW cm−2 are achieved in pseudo‐physiological buffer, artificial plasma and human plasma respectively, showing promise for in vivo or ex vivo power generation under these conditions.

Highly Efficient Flavin-Adenine Dinucleotide Glucose Dehydrogenase Fused to a Minimal Cytochrome C Domain.

Flavin-adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) is a thermostable, oxygen insensitive redox enzyme used in bioelectrochemical applications. The FAD cofactor of the enzyme is buried within the proteinaceous matrix of the enzyme, which makes it almost unreachable for a direct communication with an electrode. In this study, FAD dependent glucose dehydrogenase was fused to a natural minimal cytochrome domain in its c-terminus to achieve direct electron transfer. We introduce a fusion enzyme that can communicate with an electrode directly, without the use of a mediator molecule. The new fusion enzyme, with its direct electron transfer abilities displays superior activity to that of the native enzyme, with a kcat that is ca. 3 times higher than that of the native enzyme, a kcat/KM that is more than 3 times higher than that of GDH and 5 to 7 times higher catalytic currents with an onset potential of ca. (-) 0.15 V vs Ag/AgCl, affording higher glucose sensing selectivity. Taking these parameters into consideration, the fusion enzyme presented can serve as a good candidate for blood glucose monitoring and for other glucose based bioelectrochemical systems.

Structural analysis of fungus-derived FAD glucose dehydrogenase.

We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.

Evaluation of accuracy of FAD-GDH- and mutant Q-GDH-based blood glucose monitors in multi-patient populations

BackgroundGlucose dehydrogenases have been highly promoted to high-accuracy blood glucose (BG) monitors. The flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH) and mutant variant of quinoprotein glucose dehydrogenase (Mut. Q-GDH) are widely used in high-performance BG monitors for multi-patient use. Therefore we conducted accuracy evaluation of the GDH monitors, FAD-GDH-based GM700 and Mut. Q-GDH-based Performa. MethodsDifferent patients were enrolled: patients with and without diabetes, patients receiving respiratory therapies, hemodialysis (HD) and peritoneal dialysis (PD) patients, and neonates. The accuracy evaluation of FAD-GDH- and Mut. Q-GDH-based monitors referred to ISO 15197:2013 which applies new criteria for the minion accuracy requirements: more than 95% of the blood glucose readings shall fall within ±15mg/dL of the reference method at glucose concentration <100mg/dL and within ±15% of the reference method at glucose concentration ≥100mg/dL. Bland–Altman plots were used to evaluate the 2 GDH monitors as well. ResultsBland–Altman plots visualized excellent precision of the BG monitors. The 95% limit agreement of overall results for the FAD-GDH-based monitors was within ±12% and that for the Mut. Q-GDH-based monitors was from −10 to +17%. Both BG monitors met the accuracy requirements of ISO 15197:2013. The FAD-GDH-based monitor performed better with neonates and patients with and without diabetes, and the Mut. Q-GDH-based monitor performed better with HD and PD patients. ConclusionsAnalytical results prove that the GDH-based monitors tolerate a broad BG concentration range, are oxygen independent, have BG specificity, and have minimal interference from hematocrit. The GDH-based monitors are reliable for multi-patient use.