Phenazine Methosulfate Research Articles

Determination of Superoxide Radical Scavenging Activity and Total Antioxidant Capacity by Modified DMPD Method

Among the reactive oxygen species, Superoxide radicals can produce dangerous species that cause lipid peroxidation. Therefore, the determination and scavenging of superoxide radicals is critical. Our study is based on the interaction of the superoxide radical produced from the β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate and phenazine methosulfate (NADH-PMS) system with N, N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) to form the pink colored DMPD-quinone (DMPDQ) radical. In the presence of scavengers with superoxide radical scavenging activity (antioxidants, herbal teas) the color intensity decreases due to reduced DMPDQ radical production. The absorbance of the colored reference solution and the sample solution containing the radical scavenger was measured at 552 nm. The difference in absorbance (ΔA) between the reference solution and the sample solution was found. ΔA is proportional to the scavenger concentration. In the study, the superoxide radical scavenging effect of trolox (TR) and different AOXs was investigated. The superoxide radical scavenging effect of three different herbal tea infusion solutions was measured with this method. From the graph drawn between herbal tea concentrations and percentage inhibition values, 50% inhibition (EC50) values of herbal teas were found. EC50 method values were compared with the EC50 values of the nitroblue tetrazolium (NBT) and the 2,2'-azino-bis(3-etilbenzotiyazolin-6-sülfonik asit (ABTS) method. In addition, ABTS, cupric reducing antioxidant capacity (CUPRAC), and this study total antioxidant capacity (TAC) values of herbal tea infusions were calculated and compared.

Redox mediator interaction with Cupriavidus necator – spectroelectrochemical online analysis

Bioelectrochemical systems with Cupriavidus necator present a viable solution for harnessing H2/CO2 mixtures as substrates, employing mediated electron transfer to an infinite electron acceptor in the form of an anode instead of O2. Fourteen redox mediators were spectroelectrochemically characterized, and their efficiency was evaluated through screening with C. necator in common cuvettes with screen printed electrodes (e-Cuvettes). Key performance indicators, including total turnover number, reduction rate, and growth, were analyzed. Ferricyanide emerged as highly effective for anodic respiration, reaching a total turnover number of 8.38 over 120 h of cultivation. On the other hand, phenazine methosulfate exhibited the highest reduction rate at 2.49 mM h−1 with a total of 5.16 turnovers. Contrary, growth impairment is reported for menadione, possibly leading to deficient anodic electron transfer. The utilization of a broad spectrum of these shuttle molecules highlights the potential for optimizing bioelectrochemical applications involving C. necator.

Development of superoxide dismutase based visual and spectrophotometric method for rapid differentiation of fresh and frozen-thawed buffalo meat

Study aimed to develop biomarker-based assay for rapid detection of fresh and frozen-thawed buffalo meat in the supply chain. The method is based on development of a solvent system and identification of suitable substrate and developer for screening of biomarkers. For the confirmation column chromatography, gel electrophoresis and Western Blotting were carried out. Validation was done by intra- and inter-day validation, storability study, and determination of thermal history. Best results were shown with pH 8.0 Tris-HCl; extraction buffer, 205 µM nicotinamide adenine dinucleotide hydrogen; substrate, 184 µM Nitroblue tetrazolium, and 1.9 µM phenazine methosulfate; developer. The thermal history ranged from 0.14 to 0.17 during storage at −20 °C. The intra- and inter-day assay precision (CV %) ranged from 5.3 to 6.5 %; in chilled and 14.1 – 9.2 % in frozen-thawed samples. The study confirmed SOD as a viable biomarker. Developed method using SOD has significant potential for rapidly differentiating chilled or frozen-thawed meat.

Single-molecule electrochemical imaging of 'split waves' in the electrocatalytic (EC') mechanism.

We describe a single-molecule electrochemical imaging strategy to study the electrocatalytic (EC') mechanism. Using the fluorescent molecule ATTO647N at extremely low concentrations as the substrate, we confirmed its catalytic reduction to a nonfluorescent form in the presence of the mediator phenazine methosulfate (PMS) by imaging and counting fluorescent molecules. Conventional electrochemical current in cyclic voltammetry would not have allowed us to infer the existence of an EC' process or the PMS-mediated ATTO647N reduction. Additionally, we observed shifts in the catalytic reduction potential of ATTO647N at various mediator concentrations, which agree with the theoretical predictions by Savéant. Our work offers a new perspective on connecting single-molecule EC' behaviors with the conventional ensemble EC' mechanism, both practically and theoretically.

Identification and application of a novel deoxynivalenol-degrading enzyme from Youhaiella tibetensis

Deoxynivalenol (DON) poses a significant threat to human health due to its widespread distribution and biological toxicity. Here, we identified a novel DON-degrading enzyme from Youhaiella tibetensis (YoDDH). YoDDH exhibited the highest activity against DON at pH 4.5 and 40 ℃, in the presence of Ca2+ and the pyrroloquinoline quinone (PQQ). Additionally, YoDDH displayed remarkable thermostability at 40 ℃, with a half-life of 24 h and a Tm value of 48.5 ℃. Notably, phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP) can also serve as electron acceptors for YoDDH. After incubation in the optimal conditions for 3 h, YoDDH degraded 73 % of DON (100 μM) finally. The kcat and kcat /Km of YoDDH towards DON was determined as 1.65 s−1 and 1526 M−1·s−1 in the presence of PMS. The 3-keto-DON was verified as the degradation product. This identified YoDDH presents a promising candidate for DON decontamination in the food and feed industry.

The Reversible Electrochemical Interconversion of Formate and CO2 by Formate Dehydrogenase from Cupriavidus necator.

The bacterial molybdenum (Mo)-containing formate dehydrogenase (FdsDABG) from Cupriavidus necator is a soluble NAD+-dependent enzyme belonging to the DMSO reductase family. The holoenzyme is complex and possesses nine redox-active cofactors including a bis(molybdopterin guanine dinucleotide) (bis-MGD) active site, seven iron-sulfur clusters, and 1 equiv of flavin mononucleotide (FMN). FdsDABG catalyzes the two-electron oxidation of HCOO- (formate) to CO2 and reversibly reduces CO2 to HCOO- under physiological conditions close to its thermodynamic redox potential. Here we develop an electrocatalytically active formate oxidation/CO2 reduction system by immobilizing FdsDABG on a glassy carbon electrode in the presence of coadsorbents such as chitosan and glutaraldehyde. The reversible enzymatic interconversion between HCOO- and CO2 by FdsDABG has been realized with cyclic voltammetry using a range of artificial electron transfer mediators, with methylene blue (MB) and phenazine methosulfate (PMS) being particularly effective as electron acceptors for FdsDABG in formate oxidation. Methyl viologen (MV) acts as both an electron acceptor (MV2+) in formate oxidation and an electron donor (MV+•) for CO2 reduction. The catalytic voltammetry was reproduced by electrochemical simulation across a range of sweep rates and concentrations of formate and mediators to provide new insights into the kinetics of the FdsDABG catalytic mechanism.

A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources.

The detection of superoxide anion (O2●-) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O2●- since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O2●-. In O2●- producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O2●- generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO-). To exclude the effects of altered tyrosine phosphorylation, O2●- was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O2●- in biological systems.

Identification of neurotherapeutic constituents in Ocimum gratissimum with cholinesterase and mono amine oxidase inhibitory activities, using GC-MS analysis, in vitro, and in silico approaches

Neuroprotective activities of various extracts of Ocimum gratissimum (OG), have been reported, but there is paucity of information on its neurotherapeutic constituents. This study is aimed at identifying the neurotherapeutic constituents in OG leaves using in vitro assays, GC-MS chemical investigation and in silico studies including molecular docking, ensemble-based docking, molecular dynamics (MD) simulation, clustering and ADMET filtering analysis. Methanol extract of O gratissimum (MEOG) and solvent-partition (n-hexane, ethylacetate, and methanol residue fraction) of MEOG were investigated for in vitro acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and monoamine oxidase B (MAO-B) inhibition at concentration of 0.65, 12.5, 2.5, 5, and 10 mg/mL, using donepezil, phenazine methosulfate and selegiline as reference inhibitors for AChE, BChE and MAO B respectively. n-hexane solvent partition fraction was further investigated using GC-MS analysis. Identified compounds were screened against human AChE, BChE and MAO-B activities using molecular docking and molecular dynamics. The lead phytochemicals were further analysed for ADMET in silico analysis. MEOG and the 3 fractions (n-hexane, ethylacetate, and methanol residue fraction) inhibited the activities of AChE, BChE and MAO-B in a concentration-dependent manner with AChE (IC50 = 2.380, 2.022, 2.066 and 1.079 mg/mL respectively), BChE (IC50 = 2.261, 2.126, 2.630 and 1.465 mg/mL respectively) and MAO-B (IC50 = 2.345, 1.584, 2.933 and 2.935 mg/mL respectively). From the 38 GC-MS identified compounds, 7 hit compounds were further subjected to ensemble-docking, the lead phytochemicals (LP): cholestane and 3-methoxy-morphanin presented highest multiple binding tendencies to the three enzymes. Cholestane had the highest binding energies of −9.9, −9.0 and −11 kcal/mol, while 3-methoxy-morphanin presented the second-best binding energies of −9.3, −8.2 and −10.1 kcal/mol respectively. When compared with the binding pattern of reference inhibitors of the enzyme, lead phytochemicals were orientated in the catalytic sites of the enzyme and interacted with important catalytic residues. The LP-enzyme complexes were stable during the MD simulation analysis. Cholestane and 3-methoxy-morphanin presented favorable ADMET properties over several molecular descriptors and filters, with druggable properties and ability to cross the blood-brain barrier. Hence, cholestane and 3-methoxy-morphanin, in part, or in synergy with other hit phytochemicals, may be responsible for the neurotherapeutic activities of MEOG leaves.

Fluorometric biosensing of α-amylase using an artificial allosteric biosensor immobilized on nanostructured interface

Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λex 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent Kd of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.

Electrochemical performance of Paenibacillus profundus YoMME encapsulated in alginate polymer

Gram-positive bacterium Paenibacillus profundus YoMME, entrapped in an alginate polymer onto graphite paper, preserves its extracellular electron transfer capabilities. A current density of up to 30 mA m−2 was generated at an applied potential of −200 mV (vs. SHE). Fivefold higher initial current density values were recorded at applied potentials of +220 mV and +600 mV. The electrochemical behavior of the encapsulated bioelectrodes has been also explored by cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy and evaluated by the parameters of the best-fitted equivalent electric circuit model. Over time the bacteria grow and divide within the alginate matrix, which affects considerably the capacitance and the charge transfer resistance of the coating. The impedance spectra follow the dynamics of the bacterial culture development within the alginate polymer and are useful for the prediction of the bioelectrode performance. A current density of 150 mA m−2 was achieved when the alginate was functionalized by a mixture of the redox dyes thiazolyl blue (MTT) formazan and phenazine methosulfate (PMS). It is supposed that the added artificial mediators facilitate the electron transfer from the bacteria to the electrode surface by forming conductive cascade conduits through the alginate matrix.

Ecstasy metabolites and monoamine neurotransmitters upshift the Na+/K+ ATPase activity in mouse brain synaptosomes.

3,4-Methylenedioximethamphetamine (MDMA; "ecstasy") is a psychotropic drug with well-known neurotoxic effects mediated by hitherto not fully understood mechanisms. The Na+- and K+-activated adenosine 5'-triphosphatase (Na+/K+ ATPase), by maintaining the ion gradient across the cell membrane, regulates neuronal excitability. Thus, a perturbation of its function strongly impacts cell homeostasis, ultimately leading to neuronal dysfunction and death. Nevertheless, whether MDMA affects the Na+/K+ ATPase remains unknown. In this study, we used synaptosomes obtained from whole mouse brain to test the effects of MDMA, three of its major metabolites [α-methyldopamine, N-methyl-α-methyldopamine and 5-(glutathion-S-yl)-α-methyldopamine], serotonin (5-HT), dopamine, 3,4-dihydroxy-L-phenylalanine (L-Dopa) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the Na+/K+ ATPase function. A concentration-dependent increase of Na+/K+ ATPase activity was observed in synaptosomes exposed to the tested compounds (concentrations ranging from 0.0625 to 200µM). These effects were independent of protein kinases A and C activities. Nevertheless, a rescue of the compounds' effects was observed in synaptosomes pre-incubated with the antioxidant N-acetylcysteine (1mM), suggesting a role for reactive species-regulated pathways on the Na+/K+ ATPase effects. In agreement with this hypothesis, a similar increase in the pump activity was found in synaptosomes exposed to the chemical generator of superoxide radicals, phenazine methosulfate (1-250µM). This study demonstrates the ability of MDMA metabolites, monoamine neurotransmitters, L-Dopa and DOPAC to alter the Na+/K+ ATPase function. This could represent a yet unknown mechanism of action of MDMA and its metabolites in the brain.

Fluorescent sensor based on pyrroloquinoline quinone (PQQ)-glucose dehydrogenase for glucose detection with smartphone-adapted signal analysis.

A new nano-structured platform for fluorescent analysis using PQQ-dependent glucose dehydrogenase (PQQ-GDH) was developed, particularly using a smartphone for transduction and quantification of optical signals. The PQQ-GDH enzyme was immobilized on SiO2 nanoparticles deposited on glass microfiber filter paper, providing a high load of the biocatalytic enzyme. The platform was tested and optimized for glucose determination using a wild type of the PQQ-GDH enzyme. The analysis was based on the fluorescence generated by the reduced form of phenazine methosulfate produced stoichiometrically to the glucose concentration. The fluorescent signals were generated at separate analytical spots on the paper support under wavelength (365nm) UV excitation. The images of the analytical spots, dependent on the glucose concentration, were obtained using a photo camera of a standard smartphone. Then, the images were processed and quantified using software installed in a smartphone. The developed biocatalytic platform is the first step to assembling a large variety of biosensors using the same platform functionalized with artificial allosteric chimeric PQQ-dependent glucose dehydrogenase activated with different analytes. The future combination of the artificial enzymes, the presently developed analytical platform, and signal processing with a smartphone will lead to novel point-of-care and end-user biosensors applicableto virtually all possible analytes.

Development and Practical Application of Glucose Biosensor Based on Dendritic Gold Nanostructures Modified by Conducting Polymers.

In this study, graphite rod (GR) electrodes were electrochemically modified by dendritic gold nanostructures (DGNs) followed by immobilization of glucose oxidase (GOx) in the presence of mediator phenazine methosulfate (PMS). Modified with polyaniline (PANI) or polypyrrole (Ppy), GOx/DGNs/GR electrodes were used in glucose biosensor design. Different electrochemical methods were applied for the registration of glucose concentration, and constant potential amperometry (CPA) was chosen as the best one. PANI and Ppy layers synthesized enzymatically on the GOx/DGNs/GR electrodes extended the linear glucose determination range, the width of which depended on the duration of PANI- and Ppy-layers formation. Enzymatically formed polypyrrole was determined as the most suitable polymer for the modification and formation of the glucose biosensor instead of polyaniline, because it was 1.35 times more sensitive and had a 2.57 times lower limit of detection (LOD). The developed glucose biosensor based on the Ppy/GOx/DGNs/GR electrode was characterized by appropriate sensitivity (59.4 μA mM−1 cm−2), low LOD (0.070 mmol L−1), wide linear glucose determination range (up to 19.9 mmol L−1), good repeatability (8.01%), and appropriate storage stability (33 days). The performance of the developed glucose biosensor was tested in biological samples and beverages.

Protective effects of small heat shock proteins in Daphnia magna against heavy metal exposure

Daphnia magna is one of the most commonly used model organisms to assess toxicity of heavy metal and other xenobiotics. However, the lack of knowledge about important stress-resistant molecules limits our understanding of the alteration of phenotypic and physiological traits of D. magna upon stress exposures. In this study, we focused on a chaperone family of small heat shock protein (sHSP) that has been found in archaea, bacteria and eukaryotes and plays an important role in stress tolerance. A total of eleven sHSP genes (termed DmsHSP1 - DmsHSP11) were identified from the D. magna genome, whose expression profiles during exposure to heavy metal (Cd2+, Cu2+ and Zn2+) and a few other potential pollutants were evaluated via qRT-PCR and RNA-Seq analysis. The results highlighted the predominant role of DmsHSP1 with the highest basal expression level in adults and robust upregulation upon exposure to heavy metals (Cu2+ > Cd2+ > Zn2+). In vivo, recombinant protein rDmsHSP1–21 and rDmsHSP11–12.8 could not only prevent model substrates agglutination induced by heavy metals or reducer dithiotreitol (DTT), but also protect tissue proteins and enzymes from denaturation and inactivation caused by heavy metals or high temperature. Ectopically expression of DmsHSP1–21 or DmsHSP11–12.8 in E. coli conferred host enhanced resistance against various abiotic stresses including Cd2+, Cu2+ and phenazine methosulfate (PMS). Knockdown of DmsHSP1–21 by RNAi, but not for DmsHSP11–12.8, significantly increased the vulnerability of D. magna to heavy metal exposure. Our work provides systematic information on the evolution and function of sHSPs in D. magna and leads to important insights into the mechanisms by which D. magna survive in adverse environments.

Reprint of: Intracellular Production of Superoxide Radical and of Hydrogen Peroxide by Redox Active Compounds

Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of O2− and H2O2. One indication of this electron-shunting action was increased cyanide-resistant respiration and one cellular response was increased biosynthesis of the manganese-containing superoxide dismutase and of catalase. Blocking cytochrome oxidase with cyanide or azide increased the electron flow available for reduction of paraquat and presumably of the other exogenous compounds tested and thus increased their biological effects. Paraquat, pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, plumbagin, methylene blue, and azure C were all effective in elevating intracellular production of O2− and H2O2. The effect of alloxan appeared paradoxical in that it increased cyanide-resistant respiration without significantly increasing the cell content of the manganese-superoxide dismutase and with only a small effect on the level of catalase. The alloxan effect on cyanide-resistant respiration was artifactual and was due to an oxygen-consuming reaction between alloxan and cyanide, rather than to a diversion of the intracellular electron flow. With paraquat as a representative electron-shunting compound, the increase in biosynthesis of the manganese-superoxide dismutase was prevented by inhibitors of transcription or of translation, but not by an inhibitor of replication. The increase in this enzyme activity, caused by paraquat and presumably by the other compounds, was thus due to de novo enzyme synthesis activated or derepressed at the level of transcription.

A quinoprotein dehydrogenase from Pelagibacterium halotolerans ANSP101 oxidizes deoxynivalenol to 3-keto-deoxynivalenol

Deoxynivalenol (DON), a notorious mycotoxin commonly present in cereal grains, poses severe health risks to human and livestock. A quinoprotein from Pelagibacterium halotolerans ANSP101, named deoxynivalenol dehydrogenase (DDH), was identified by comparative genome analysis of P. halotolerans ANSP101, Devosia mutan 17-2-E−8 and Devosia strain IFO13580. DDH was responsible for oxidation of DON into 3-keto-DON with hydrogen acceptor phenazine methosulfate (PMS) or dichlorophenolindophenol (DCPIP). DDH had two catalytic amino acid residues (Ser478, Glu480), and 57.55% sequence identity with quinoprotein dehydrogenases DepA from Devosia sp. 17-2-E−8 and QDDH from Devosia sp. D6-9 by sequence alignment. The Met516 of DDH was changed to Glu516 by site-specific mutagenesis, and the new mutant was designated as TDDH. The mutant TDDH possessing catalytic triplet SEE (Ser478, Glu480, Glu516) could degrade DON to 3-keto-DON with pyrroloquinoline quinone (PQQ) as hydrogen acceptor besides PMS and DCPIP. Importantly, the mutant TDDH exhibited stronger degradation ability for DON in the presence of the same hydrogen acceptor compared with wild DDH. The mutant TDDH had an effect on degrading DON with PQQ at a wide range of pH (6.0–11.0) and temperatures (20–45 °C). The results provide potential use of TDDH as a detoxification agent to mitigate DON hazard on human and animals applied in food and feed.

Nanostructured Interface Loaded with Chimeric Enzymes for Fluorimetric Quantification of Cyclosporine A and FK506.

Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.

The Impact of Glucose Oxidase Immobilization on Dendritic Gold Nanostructures on the Performance of Glucose Biosensors.

In recent years, many efforts have been made to develop rapid, sensitive and user-friendly glucose biosensors for monitoring blood glucose concentration in patients. In this study, the electrochemical glucose biosensors based on graphite rod (GR) electrode electrochemically modified with dendritic gold nanostructures (DGNs) and glucose oxidase (GOx) were developed. Phenazine methosulfate was used as a soluble redox mediator. Three GOx immobilization methods: adsorption on DGNs and cross-linking with glutaraldehyde (GA) vapour (GA-GOx/DGNs/GR), covalent immobilization on DGNs modified with 11-mercaptoundecanoic acid self-assembled monolayer (SAM) (GOx-SAM/DGNs/GR) and covalent immobilization on SAM with additional cross-linking with GA vapour (GA-GOx-SAM/DGNs/GR), were used. It was determined that GA significantly improved the stability of the enzyme layer. The difference of maximal current generated during the enzymatic reaction (ΔImax) equal to 272.06 ± 8.69 µA was obtained using a biosensor based on GA-GOx/DGNs/GR electrodes. However, the highest ΔImax equal to 384.20 ± 16.06 µA was obtained using GA-GOx-SAM/DGNs/GR electrode. ΔImax for biosensors based on the GA-GOx-SAM/DGNs/GR electrode was 1.41 times higher than for the GA-GOx/DGNs/GR, whereas the linear dynamic range from 0.1 to 10 mM was the same using all three GOx immobilization methods. The limit of detection using GA-GOx-SAM/DGNs/GR and GA-GOx/DGNs/GR electrodes was 0.019 and 0.022 mM, respectively. The ability to detect glucose in the serum by developed biosensors was evaluated.

Deterministic flow cytometry: Rapid erythrocyte rheological profiling from a drop of whole blood

The loss of erythrocyte deformability (ED) is part of the etiology of a wide range of diseases. Current measures of ED are limited by the complexity of laboratory-based methods, insensitivity and the need for a venous blood draw. Here, we introduce a deterministic flow cytometry (DFC) assay for label-free measurements of the size of erythrocyte and ED. The assay can be completed within 2 min using only 10 μL whole blood collected via finger pricking, suitable for point-of-care measurements for clinical profiling of neonates, infants and children. Our results show that the unique C-shaped DFC is a sensitive and easy-to-use tool, which can detect not only the biphasic effect of ethanol and oxidative damage of erythrocytes but also significant individual-dependent variability and dose dependence under the phenazine methosulfate (PMS) stimuli. We also demonstrated the application of DFC for the assessment of sensitive erythrocyte changes in sepsis induced rats.

Characterization of a sorbose oxidase involved in the biosynthesis of 2-keto-L-gulonic acid from Gluconobacter oxydans WSH-004

2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, can be synthesized from L-sorbose by many microorganisms via “sorbose dehydrogenase” and “sorbosone dehydrogenase (SNDH)”. However, only a few studies were carried out on the catalytic properties of sorbose dehydrogenase in Gluconobacter oxydans. In this study, sorbose dehydrogenase of G. oxydans WSH-004 was identified. Different from previous findings, this enzyme turned out to be a sorbose oxidase (SOX) rather than a sorbose dehydrogenase·H2O2 was determined in the oxidization of L-sorbose to L-sorbosone and it also showed inhibition on the activity of SOX. Phenazine methosulfate (PMS) was the best electron acceptor of SOX and the Km of SOX for L-sorbose was 117 mM when dichlorophenolindophenol (DCIP) was used as the electron acceptor. Finally, the enzymatic catalysis of SOX and SNDH was conducted in vitro. 2-KLG could be produced from L-sorbose by SOX and SNDH with nicotinamide adenine dinucleotide phosphate (NADP+) as a cofactor. These results were useful in the reconstruction of G. oxydans to synthesize 2-KLG in a one-step fermentation process.