Enhancing levan biosynthesis by destroying the strongly acidic environment caused by membrane-bound glucose dehydrogenase (mGDH) in Gluconobacter sp. MP2116.

Levan produced by Gluconobacter spp. has great potential in biotechnological applications. However, Gluconobacter spp. can synthesize organic acids during fermentation, resulting in environmental acidification. Few studies have focused on the effects of environmental acidification on levan synthesis. This study revealed that the organic acids, mainly gluconic acid (GA) and 2-keto-gluconic acid (2KGA) secreted by Gluconobacter sp. MP2116 created a highly acidic environment (pH < 3) that inhibited levan biosynthesis. The levansucrase derived from strain MP2116 had high enzyme activity at pH 4.0 âˆ¼ pH 6.5. When the ambient pH was less than 3, the enzyme activity decreased by 67 %. Knocking out the mgdh gene of membrane-bound glucose dehydrogenase (mGDH) in the GA and 2KGA synthesis pathway in strain MP2116 eliminated the inhibitory effect of high acid levels on levansucrase function. As a result, the levan yield increased from 7.4 g/l (wild-type) to 18.8 g/l (Δmgdh) during fermentation without pH control. This study provides a new strategy for improving levan production by preventing the inhibition of polysaccharide synthesis by environmental acidification.

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

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

How carbon sources drive cellulose synthesis in two Komagataeibacter xylinus strains.

Bacterial cellulose synthesis from defined media and waste products has attracted increasing interest in the circular economy context for sustainable productions. In this study, a glucose dehydrogenase-deficient Δgdh K2G30 strain of Komagataeibacter xylinus was obtained from the parental wild type through homologous recombination. Both strains were grown in defined substrates and cheese whey as an agri-food waste to assess the effect of gene silencing on bacterial cellulose synthesis and carbon source metabolism. Wild type K2G30 boasted higher bacterial cellulose yields when grown in ethanol-based medium and cheese whey, although showing an overall higher D-gluconic acid synthesis. Conversely, the mutant Δgdh strain preferred D-fructose, D-mannitol, and glycerol to boost bacterial cellulose production, while displaying higher substrate consumption rates and a lower D-gluconic acid synthesis. This study provides an in-depth investigation of two K. xylinus strains, unravelling their suitability for scale-up BC production.

Effects of Cross-linker Chemistry on Bioelectrocatalytic Reactions in a Redox Cross-linked Network of Glucose Dehydrogenase and Thionine.

Enzyme-mediator bioconjugation is emerging as a building block for designing electrode platforms for the construction of biosensors and biofuel cells. Here, we report a one-pot bioconjugation technique for flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and thionine (TH) using a series of cross-linkers, including epoxy, N-hydroxysuccinimide (NHS), and aldehydes. In this technique, FAD-GDH and thionine are conjugated through an amine cross-linking reaction to generate a redox network, which has been successfully employed for the oxidation of glucose. The bioconjugation chemistry of cross-linkers with the amino groups on FAD-GDH and thionine plays a vital role in generating distinct network structures. The epoxy-type cross-linker reacts with the primary and secondary amines of thionine at room temperature, thereby producing an FAD-GDH-TH-FAD-GDH hyperbranched bioconjugate network, the aldehyde undergoes a rapid cross-linking reaction to produce a network of FAD-GDH-FAD-GDH, while the NHS-based cross-linker can react with the primary amines of both FAD-GDH and thionine, forming an FAD-GDH-cross-linker-TH polymeric network. This reaction has the potential to enable the conjugation of a redox mediator with a FAD-GDH network, which is particularly essential when designing an enzyme electrode platform. The data demonstrated that the polymeric cross-linked network based on the NHS cross-linker exhibited a considerable increase in electron transport while producing a catalytic current of 830 µA cm-2. The cross-linker spacer arm length also affects the overall electrochemical function of the network and its performance; an adequate spacer length containing a cross-linker is required, resulting in a faster electron transfer. Finally, a leaching test confirmed that the stability of the enzyme electrode was improved when the electrode was tested using the redox probe. This study elucidates the relationship between cross-linking chemistry and redox network structure and enhances the high performance of enzyme electrode platforms for the oxidation of glucose.

Enzymatic Precipitation of Highly Electroactive and Ion-Transporting Prussian Blue for a Sensitive Electrochemical Immunosensor.

Sensitive and/or multiplex electrochemical biosensors often require efficient (bio)catalytic conversion of substrates into insoluble electroactive products. The enzymatic formation and precipitation of coordination polymers under mild conditions offers a promising solution for this purpose. Herein, we report the enzymatic precipitation of Prussian blue (PB), a highly electroactive and ion-transporting coordination polymer, on an immunosensing electrode for application in a sensitive electrochemical immunosensor for detecting thyroid-stimulating hormone (TSH). Five pairs of redox enzymes and their specific reductants were examined to achieve rapid PB precipitation and electrochemical oxidation. Among these pairs, O2-insensitive flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) paired with glucose yielded the highest electrochemical signal-to-background (S/B) ratio. FAD-GDH catalyzed the conversion of Fe(CN)63- to Fe(CN)64-, which coordinated with Fe3+, leading to PB formation and subsequent precipitation through repeated conversions. The resulting PB precipitate, with its close proximity to the electrode, facilitated rapid electrochemical oxidation and generated a strong electrochemical signal. Notably, the precipitation and electrochemical oxidation of PB were more effective than those of its analogues. When applied to a sandwich-type immunosensor for TSH detection, the enzymatic PB precipitation achieved a calculated detection limit of approximately 2 pg/mL in artificial serum, covering the clinically relevant range. These findings indicate the potential widespread utility of PB precipitation and electrochemical oxidation for sensitive multiplex biomarker detection.

Does Acinetobacter calcoaceticus glucose dehydrogenase produce self-damaging H2O2?

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

Enhancing Glucose Biosensing with Graphene Oxide and Ferrocene-Modified Linear Poly(ethylenimine).

We designed and optimized a glucose biosensor system based on a screen-printed electrode modified with the NAD-GDH enzyme. To enhance the electroactive surface area and improve the electron transfer efficiency, we introduced graphene oxide (GO) and ferrocene-modified linear poly(ethylenimine) (LPEI-Fc) onto the biosensor surface. This strategic modification exploits the electrostatic interaction between graphene oxide, which possesses a negative charge, and LPEI-Fc, which is positively charged. This interaction results in increased catalytic current during glucose oxidation and helps improve the overall glucose detection sensitivity by amperometry. We integrated the developed glucose sensor into a flow injection (FI) system. This integration facilitates a swift and reproducible detection of glucose, and it also mitigates the risk of contamination during the analyses. The incorporation of an FI system improves the efficiency of the biosensor, ensuring precise and reliable results in a short time. The proposed sensor was operated at a constant applied potential of 0.35 V. After optimizing the system, a linear calibration curve was obtained for the concentration range of 1.0-40 mM (R2 = 0.986). The FI system was successfully applied to determine the glucose content of a commercial sports drink.

UDP-6-glucose dehydrogenase in hormonally responsive breast cancers.

Survival for metastatic breast cancer is low and thus, continued efforts to treat and prevent metastatic progression are critical. Estrogen is shown to promote aggressive phenotypes in multiple cancer models irrespective of estrogen receptor (ER) status. Similarly, UDP-Glucose 6-dehydrogenase (UGDH) a ubiquitously expressed enzyme involved in extracellular matrix precursors, as well as hormone processing increases migratory and invasive properties in cancer models. While the role of UGDH in cellular migration is defined, how it intersects with and impacts hormone signaling pathways associated with tumor progression in metastatic breast cancer has not been explored. Here we demonstrate that UGDH knockdown blunts estrogen-induced tumorigenic phenotypes (migration and colony formation) in ER+ and ER- breast cancer in vitro. Knockdown of UGDH also inhibits extravasation of ER- breast cancer ex vivo, primary tumor growth and animal survival in vivo in both ER+ and ER- breast cancer. We also use single cell RNA-sequencing to demonstrate that our findings translate to a human breast cancer clinical specimen. Our findings support the role of estrogen and UGDH in breast cancer progression provide a foundation for future studies to evaluate the role of UGDH in therapeutic resistance to improve outcomes and survival for breast cancer patients.

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.

UDP-glucose dehydrogenase supports autophagy-deficient PDAC growth via increasing hyaluronic acid biosynthesis.

Autophagy is an essential degradation and recycling process that maintains cellular homeostasis during stress or nutrient deprivation. However, certain types of tumors such as pancreatic cancers can circumvent autophagy inhibition to sustain growth. The mechanism that autophagy-deficient pancreatic ductal adenocarcinoma (PDAC) uses to grow under nutrient deprivation is poorly understood. Our data show that nutrient deprivation in PDAC results in UDP-glucose dehydrogenase (UGDH) degradation, which is dependent on autophagic cargo receptor sequestosome 1 (p62). Moreover, we demonstrate that accumulated UGDH is indispensable for autophagy-deficient PDAC cells proliferation by promoting hyaluronic acid (HA) synthesis upon energy deprivation. Using an orthotopic mouse model of PDAC, we find that inhibition of HA synthesis by targeting UGDH in PDAC reduces tumor weight. Thus, the combined inhibition of HA and autophagy might be an attractive strategy for PDAC treatment.

The application of bacteria-derived dehydrogenases and oxidases in the synthesis of gold nanoparticles.

In this work the green synthesis of gold nanoparticles (Au-NPs) using the oxidoreductive enzymes Myriococcum thermophilum cellobiose dehydrogenase (Mt CDH), Glomerella cingulata glucose dehydrogenase (Gc GDH), and Aspergillus niger glucose oxidase (An GOX)) as bioreductants was investigated. The influence of reaction conditions on the synthesis of Au-NPs was examined and optimised. The reaction kinetics and the influence of Au ions on the reaction rate were determined. Based on the kinetic study, the mechanism of Au-NP synthesis was proposed. The Au-NPs were characterized by UV-Vis spectroscopy and transmission electron microscopy (TEM). The surface plasmon resonance (SPR) absorption peaks of the Au-NPs synthesised with Mt CDH and Gc GDH were observed at 535 nm, indicating an average size of around 50 nm. According to the image analysis performed on a TEM micrograph, the Au-NPs synthesized with Gc GDH have a spherical shape with an average size of 2.83 and 6.63 nm after 24 and 48 h of the reaction, respectively. KEY POINTS: • The Au NPs were synthesised by the action of enzymes CDH and GDH. • The synthesis of Au-NPs by CDH is related to the oxidation of cellobiose. • The synthesis of Au-NPs by GDH was not driven by the reaction kinetic.

Reduced Graphene Oxide/Organic Dye Composites for Bioelectroconversion of Saccharides: Application for Detection of Saccharides and α-Amylase Assessments.

In this study, PQQ-dependent glucose dehydrogenase (PQQ-GDH) was immobilized onto reduced graphene oxide (rGO) modified with organic dyes from three different classes (acridine, arylmethane, and diazo); namely, neutral red (NR), malachite green (MG), and congo red (CR) formed three types of biosensors. All three rGO/organic dye composites were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. The impact of three rGO/organic dye modifications employed in bioelectrocatalytic systems on changes in enzyme activity and substrate selectivity was investigated. The highest sensitivity of 39 µA/cm2 was obtained for 1 mM of glucose when a rGO_MG/PQQ-GDH biosensor was used. A significant improvement in the electrochemical response of biosensors was attributed to the higher amount of pyrrolic nitrogen groups on the surface of the rGO/organic dye composites. Modifications of rGO by NR and MG not only improved the surfaces for efficient direct electron transfer (DET) but also influenced the enzyme selectivity through proper binding and orientation of the enzyme. The accuracy of the biosensor's action was confirmed by the spectrophotometric analysis. Perspectives for using the proposed bioelectrocatalytic systems operating on DET principles for total or single monosaccharide and/or disaccharide determination/bioconversion systems or for diagnoses have been presented through examples of bioconversion of D-glucose, D-xylose, and maltose.

System Accuracy and Interference Evaluation of a New Glucose Dehydrogenase-Based Blood Glucose Meter for Patient Self-Testing.

New European medical device regulations require the performance of postmarketing surveillance evaluations for blood glucose meters (BGMs). We conducted an ISO15197:2015-conform system performance evaluation with the approved glucose dehydrogenase (GDH)-based Wellion NEWTON BGM. One hundred subjects were enrolled into the study (44 female, 56 male, 43 healthy subjects, 23 type 1 diabetes, 34 type 2 diabetes, age: 53.7 ± 15.8 years). In addition, manipulated heparinized whole blood was used for a laboratory interference test with ten selected substances (interference definition: substance-induced bias > 10%). The mean absolute relative difference (MARD) was 4.7%, and 100% of the values were in zones A (99.7%) and B (0.3%), respectively, of the consensus error grid. Interference was observed with xylose only, which is a known interfering substance for GDH-based BGMs.

UDP-glucose dehydrogenase (UGDH) in clinical oncology and cancer biology.

UDP-glucose-6-dehydrogenase (UGDH) is a cytosolic, hexameric enzyme that converts UDP-glucose to UDP-glucuronic acid (UDP-GlcUA), a key reaction in hormone and xenobiotic metabolism and in the production of extracellular matrix precursors. In this review, we classify UGDH as a molecular indicator of tumor progression in multiple cancer types, describe its involvement in key canonical cancer signaling pathways, and identify methods to inhibit UGDH, its substrates, and its downstream products. As such, we position UGDH as an enzyme to be exploited as a potential prognostication marker in oncology and a therapeutic target in cancer biology.

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system.

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.

Construction of metal-organic framework-based multienzyme system for L-tert-leucine production.

Chiral compounds are important drug intermediates that play a critical role in human life. Herein, we report a facile method to prepare multi-enzyme nano-devices with high catalytic activity and stability. The self-assemble molecular binders SpyCatcher and SpyTag were fused with leucine dehydrogenase and glucose dehydrogenase to produce sc-LeuDH (SpyCatcher-fused leucine dehydrogenase) and GDH-st (SpyTag-fused glucose dehydrogenase), respectively. After assembling, the cross-linked enzymes LeuDH-GDH were formed. The crosslinking enzyme has good pH stability and temperature stability. The coenzyme cycle constant of LeuDH-GDH was always higher than that of free double enzymes. The yield of L-tert-leucine synthesis by LeuDH-GDH was 0.47 times higher than that by free LeuDH and GDH. To further improve the enzyme performance, the cross-linked LeuDH-GDH was immobilized on zeolite imidazolate framework-8 (ZIF-8) via bionic mineralization, forming LeuDH-GDH @ZIF-8. The created co-immobilized enzymes showed even better pH stability and temperature stability than the cross-linked enzymes, and LeuDH-GDH@ZIF-8 retains 70% relative conversion rate in the first four reuses. In addition, the yield of LeuDH-GDH@ZIF-8 was 0.62 times higher than that of LeuDH-GDH, and 1.38 times higher than that of free double enzyme system. This work provides a novel method for developing multi-enzyme nano-device, and the ease of operation of this method is appealing for the construction of other multi-enzymes @MOF systems for the applications in the kinds of complex environment.

Glucose Dehydrogenases-Mediated Acclimation of an Important Rice Pest to Global Warming.

Global warming is posing a threat to animals. As a large group of widely distributed poikilothermal animals, insects are liable to heat stress. How insects deal with heat stress is worth highlighting. Acclimation may improve the heat tolerance of insects, but the underlying mechanism remains vague. In this study, the high temperature of 39 °C was used to select the third instar larvae of the rice leaf folder Cnaphalocrocis medinalis, an important insect pest of rice, for successive generations to establish the heat-acclimated strain (HA39). The molecular mechanism of heat acclimation was explored using this strain. The HA39 larvae showed stronger tolerance to 43 °C than the unacclimated strain (HA27) persistently reared at 27 °C. The HA39 larvae upregulated a glucose dehydrogenase gene, CmGMC10, to decrease the reactive oxygen species (ROS) level and increase the survival rate under heat stress. The HA39 larvae maintained a higher activity of antioxidases than the HA27 when confronted with an exogenous oxidant. Heat acclimation decreased the H2O2 level in larvae under heat stress which was associated with the upregulation of CmGMC10. The rice leaf folder larvae may acclimate to global warming via upregulating CmGMC10 to increase the activity of antioxidases and alleviate the oxidative damage of heat stress.

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

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

Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+ ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA+ . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Direct Electrochemistry of Glucose Dehydrogenase-Functionalized Polymers on a Modified Glassy Carbon Electrode and Its Molecular Recognition of Glucose.

A glucose biosensor was layer-by-layer assembled on a modified glassy carbon electrode (GCE) from a nanocomposite of NAD(P)+-dependent glucose dehydrogenase, aminated polyethylene glycol (mPEG), carboxylic acid-functionalized multi-wall carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymers. The electrochemical electrode was denoted as NF/IL/GDH/mPEG-fMWCNTs/GCE. The composite polymer membranes were characterized by cyclic voltammetry, ultraviolet-visible spectrophotometry, electrochemical impedance spectroscopy, scanning electron microscopy, and transmission electron microscopy. The cyclic voltammogram of the modified electrode had a pair of well-defined quasi-reversible redox peaks with a formal potential of -61 mV (vs. Ag/AgCl) at a scan rate of 0.05 V s-1. The heterogeneous electron transfer constant (ks) of GDH on the composite functional polymer-modified GCE was 6.5 s-1. The biosensor could sensitively recognize and detect glucose linearly from 0.8 to 100 µM with a detection limit down to 0.46 µM (S/N = 3) and a sensitivity of 29.1 nA µM-1. The apparent Michaelis-Menten constant (Kmapp) of the modified electrode was 0.21 mM. The constructed electrochemical sensor was compared with the high-performance liquid chromatography method for the determination of glucose in commercially available glucose injections. The results demonstrated that the sensor was highly accurate and could be used for the rapid and quantitative determination of glucose concentration.