Preparation and in vitro evaluation of polylactic acid nanoparticles containing arginine and glucose oxidase / 药学学报

Hydrogen peroxide (H2O2) and nitric oxide (NO) has a short half-life, low bioavailability, poor tumor targeting and systemic adverse reactions in the physiological environment. In this study, phacoemulsification and nano-precipitation were used to synthesize didecyl dimethyl ammonium bromide (DDAB)/polylactic acid nanoparticles (PLA), then L-arginine (L-Arg) and glucose oxidase (GOx)-loaded nanoparticles (GADP) were prepared, and the in vitro antitumor activity was investigated．The particle size, potential, embedding rate and the ability to produce H2O2/NO of the nanoparticles were investigated. Meanwhile, in vitro cell cytotoxicity against human hepatoma cells (HepG2) was evaluated．The results showed that the prepared L-Arg-DDAB/PLA (ADP) nanoparticles were spherical particles. And the particle size and zeta potential were (225.7 ± 6.33) nm and (+23.5 ± 0.12) mV, respectively. The adsorption rate of GOx was 87.23% ± 0.02%. The drug loading of L-Arg was 15.6% ± 0.22%. The pH value of glucose solution and the amount of H2O2 showed that GADP had good catalytic activity. In vitro cytotoxicity experiments showed that blank nanoparticles were nontoxic, while the drug-loaded nanoparticles presented enhanced antitumor effect on HepG2 cells. And can inhibit tumor cell migration. The low dose nano-scale NO delivery system GADP can effectively inhibit the migration of tumor cells and kill tumor cells, thus producing therapeutic benefits.

Correlation of fasting blood, salivary glucose and malondialdehyde in subjects with & without type 2 diabetes

Oxidative stress is identified as the common pathogenic factor that leads to insulin resistance in diabetics. Malondialdehyde is a product of lipid peroxidation. Aim: The aim of this study was to determine the variation in the Salivary malondialdehyde (MDA) among subjects with and without T2DM in comparison to the fasting blood and Salivary glucose. Methods: This study involved 29 healthy participants as Controls (group I) and 29 participants with Type 2 Diabetes Mellitus as Cases (group II). Salivary Glucose was analysed by glucose oxidase end-point assay. Thiobarbituric acid (TBA) assay method was considered for estimation of MDA in fasting saliva. Data was Statistically analysed using SPSS20. Parametric test was performed to analyse the data. Results: The correlation calculated between FBG with FSG level was found to be highly significant. A positive correlation between MDA levels with FBG was found. The relationship between FBG and FSG (r = 0.7815, p < 0.05), FBG and MDA (r =0.3678, p < 0.05) and FSG and MDA (r = 0.2869, p < 0.05) were found to be positively significant. Conclusion: Saliva as a unique body fluid can serve as a medium for biochemical analysis only in standard settings and with multiple measures to be used as a diagnostic tool in par with the gold standard serum. Salivary MDA levels can be considered as one of the oxidative stress markers in Type 2 Diabetic condition

Progress of intelligent-responsive insulin delivery mediated by glucose oxidase / 中国药科大学学报

@#The delivery of exogenous insulin is very important for the treatment of type 1 and advanced type 2 diabetes.Traditional injectable administration is prone to cause hypoglycemia, while the intelligent insulin system has the advantages of safety, long-term effectiveness, and high responsiveness.Glucose oxidase (GOx) can consume O2 and catalyze glucose to produce gluconic acid and H2O2.Therefore, the O2 level, H2O2 content and pH of GOx system are closely related to the glucose level in the system.This review introduces the application of GOx in closed-loop insulin delivery systems at home and abroad in recent years, which can be divided into single response and multiple response of pH-response, hypoxia-response, and H2O2-response according to the mechanism.It also discusses the opportunities and challenge facing by the application of GOx in the future.

Anticancer therapy mediated by glucose oxidase / 肿瘤

Glucose oxidase is a kind of endogenous oxidoreductase which exi sts wi del y i n organi sm. I n recent years, due to i ts i nherent biocompatibility, low toxicity and unique catalytic effect on betaD-glucose, glucose oxidase has attracted more and more attention in the field of biomedicine. Glucose oxidase can effectively catalyze the oxidation of glucose into gluconic acid and hydrogen peroxide. Researchers have developed various anticancer therapies based on gl ucose oxi dase usi ng t hi s cat al yt i c propert y: (1) gl ucose consumption provides alternative treatment strategies for cancer starvation; (2) oxygen consumption increases tumor hypoxia, which can be used for tumor hypoxia activation therapy; (3) the production of gluconic acid increases the acidity of tumor microenvironment, which can trigger the release of pH-responsive drugs; (4) the production of hydrogen peroxide increases the oxidative stress level of tumors. Hydrogen peroxide can be converted into toxic hydroxyl radicals by being exposed to light (such as near-infrared light) or fenton reaction, thus killing cancer cells. In addition, glucose oxidase can also be used in combination with other biological enzymes, hypoxia-activated prodrugs, photosensitizers or fenton reagents to produce the multi-mode synergistic anticancer therapies with starvation therapy as the main part, cooperating with hypoxia-activated therapy, oxidation therapy, photodynamic therapy and light therapy, etc. In this review, the glucose oxidase-mediated tumor monotherapy is firstly introduced, and the multi-mode synergistic anti-cancer therapy mediated by glucose oxidase as well as its specific anti-tumor mechanism are systematically elaborated.

Prevalence and cause of gestational diabetes mellitus in a tertiary care center in Kolar district: a population based study

Background: Gestational diabetes mellitus (GDM) is carbohydrate intolerance at the onset of pregnancy which induces pathological short term or long term outcomes for both mother and baby. The aim of the present study was to know the prevalence of GDM in pregnant women who were attending the antenatal care (ANC) center at a tertiary care hospital in Kolar, Karnataka, India.Methods: This prospective study was conducted in Department of Obstetrics and Gynecology, Sri Devaraj Urs Medical College, a constituent of Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka, India. The duration of the study was two months. In this study, 108 pregnant women above 24 weeks of gestation were screened for GDM by oral glucose tolerance test. Fasting 2 milli liter blood was collected and were given 75 grams of glucose in 200 milli liters of water and asked to drink within 5 minutes. Again 2 milli liters venous blood was collected after 1 hour and 2 hours from all participants. Plasma sample was used for the estimation of glucose by glucose oxidase and peroxidase (GOD-POD) method.Results: Out of 108, 12 women (11.1%) were diagnosed with GDM. The prevalence rate was higher in the age group of 26-30 years (41.6%).  Among 12 diabetic women, five (47.2%) exercised regularly and seven (58.3%) did not doing exercise. Out of 12 GDM subjects, eight of them had family history of diabetes in first degree relatives; among which one was hypertensive and five were suffering from thyroid problems.Conclusions: In the present study, the prevalence of GDM was found to be 11.1%. Prevalence of GDM might be influenced by increasing age, pre pregnancy weight, family history of diabetes, past history of pregnancy complications, status of literacy and exercise.

In vitro study of GOD/Fe3O4 loaded PLGA nanoparticles in the treatment of retinoblastoma / 解放军医学杂志

Objective To evaluate the therapeutic potential of a novel type of Poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with glucose oxidase (GOD)/superparamagnetic iron oxide nanoparticles (Fe3O4Nps) on retinoblastoma (RB) cells in vitro. Methods PLGA nanoparticles loaded with GOD/Fe3O4 (PFG) were prepared by double emulsification. Their particle size, potential, external morphology, and internal structure were examined. Particles that made of PLGA (control), PLGA loaded with GOD (PG), and PLGA loaded with Fe3O4 (PF) are served as control. Y79 cells that were incubated with different particles are termed control group, PF group, PG group, and PFG group. After co-incubation with nanoparticles, cell viability, and reactive oxygen species production were detected. Results PLGA nanoparticles loaded with GOD/Fe3O4 were successfully prepared. The form of PLGA nanoparticles was uniform and showed a round shape with a diameter of 299.3 nm. The nanoparticles were engulfed by Y79 cells after co-incubation with Y79 cells, producing a large number of reactive oxygen species. Cytotoxicity test results showed that the cell viability of Y79 cells in PLGA nanoparticle group coated with GOD/Fe3O4 [(53.648±2.565)%] was significantly lower than that in control group [(100.028±4.491)%], PF group [(97.782±17.520)%] or PG group [(87.438±3.537)%](F=21.226, P<0.01); The cell viability of Y79 cells in 0.25 μg/ml PFG nanoparticle group [(51.770±1.529)%] was significantly lower than that in control group [(100.000±5.021)%], 0.0625 μg/ml group [(85.723±6.903)%] and 0.125 μg/ml group [(74.535±8.282)%] (F=34.593, P<0.05). Massive cell death was detected in the PFG group under laser confocal microscope. Conclusions The novel type of PLGA nanoparticles loaded with GOD/Fe3O4 toxic to Y79 tumor cells with a good reactive oxygen generation ability. It provides a potential treatment for RB.

Establecimiento de condiciones más eficientes para la producción de biosensores de glucosa / Establishment of more efficient conditions for the production of glucose biosensors

Los biosensores son dispositivos móviles que permiten detectar de forma rápida y sencilla enfermedades del metabolismo e infecciones víricas de interés veterinario y clínico, como el rotavirus y la hepatitis B y C. Este trabajo tuvo como objetivo determinar las variables significativas del proceso de producción de los biosensores de glucosa fabricados en el Centro de Inmunoensayo (La Habana, Cuba). Se produjeron ocho corridas experimentales teniendo en cuenta los procedimientos normativos de operación implementados en la planta de producción de biosensores y se realizaron las evaluaciones de calidad correspondientes (pruebas de exactitud) para liberar analíticamente los lotes producidos. Los experimentos realizados proporcionaron información acerca de cuáles variables deben controlarse con más cuidado durante la producción a fin de evitar altos niveles de productos no conformes o el comportamiento errático del proceso. Las variables seleccionadas para el estudio fueron las relacionadas con la preparación de la solución enzimática. Con los resultados obtenidos se realizó un análisis de regresión múltiple para determinar los factores estadísticamente significativos del modelo, obteniéndose un coeficiente de determinación superior al 90 por ciento, logrando explicar el 98,637 por ciento de la variación entre los valores de porcentaje de exactitud y la media. Los factores que resultaron ser significativos fueron la concentración de la enzima glucosa oxidasa, la concentración del mediador eléctrico y la conductividad del agua ultrapura para un nivel de confianza del 95 por ciento. El análisis realizado arrojó resultados satisfactorios demostrando que variando parámetros del proceso productivo es posible disminuir los valores del porcentaje de exactitud(AU)

Biosensors are mobile devices that allow rapid and easy detection of metabolic diseases and viral infections of veterinary and clinical interest, such as rotavirus and hepatitis B and C. The objective of this work was to determine the significant variables of the production process of the glucose biosensors manufactured in the Immunoassay Center (Havana, Cuba). Eight experimental runs were carried out taking into account the normative operating procedures of the biosensor production plant. Accuracy tests were carried out to release produced batches. The experiments provided information about which factors should be carefully controlled during the manufacture procedure in order to avoid high levels of faulty products or the erratic behavior of the process. The factors selected for the study were those related with the preparation of the enzymatic solution. A multiple regression analysis was carried out to determine the statistically significant factors of the model. The coefficient of determination was higher than 90 percent, and the 98.637 percent of the variation between the values of percentage of accuracy and the mean value could be explained. The significant factors were the concentration of the glucose oxidase enzyme, the electric mediator concentration, and the ultrapure water conductivity (95 percent confidence level). The analysis carried out showed satisfactory results. In the present study, it was demonstrated that varying parameters of the production process it is possible to decrease the accuracy percentage values(AU)

Enhancing thermal stability of glucose oxidase by fusing amphiphilic short peptide / 生物工程学报

Glucose oxidase catalyzes the oxidation of β-D-glucose to gluconic acid and its derivatives, thus shows a great potential in the development of antibiotic-free feed. However, its production and processing still have the problem of poor thermal stability of enzyme activity. In this study, fusion of amphiphilic peptide technology was used to improve the stability of glucose oxidase. Herein, eight self-assembling peptides with different amino acid lengths and Linkers were fused to the N terminus of the glucose oxidase, yielding eight chimeric fusions SAP1-GS-GOD, SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD, SAP4-PT-GOD, SAP5-PT-GOD, SAP6-PT-GOD and SAP7-PT-GOD. Then, the 8 recombinant proteins were expressed in P. pastoris GS115. After separation and purification, the stability of glucose oxidase at 60 ℃was determined. The relative enzyme activities of the PT Linker-linked fusion enzyme incubated at 60 ℃ for 60 min were higher than those of the original enzyme, and the relative activity of SAP5-PT-GOD was 67% at 60 ℃ for 30 min, which was 10.9 times higher than that of the initial enzyme with the same treatment. Among them, the Kcat/Km value of SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD and SAP5-PT-GOD of the fusion enzyme was further improved than that of the initial enzyme. Through the analysis of the intramolecular force of the fusion enzyme, the increase of the thermal stability of the fusion enzyme is mainly due to the increase of the hydrogen bond. In summary, the study indicates that translational fusion of self-assembling peptides with PT Linker was able to augment the thermo-stability of glucose oxidase, which has certain potential in the production and application of glucose oxidase. The glucose oxidase with improved thermostability obtained in the above study and the related mechanism will play an important role in improving the activity of related enzymes in the proceeding of processing and application.

Study of antioxidant effect of cannabinoid receptor type 2 agonist on rat hepatic stellate cell line / 中华肝脏病杂志

Objective@#To investigate the action and antioxidant effects of CB2 agonist AM-1241 on rat hepatic stellate cell line (HSC-T6).@*Methods@#HSC-T6 was randomly divided into four groups: control group, oxidative stress group, AM-1241 intervention group and AM-1241+AM-630 antagonist group. Survival rate of HSC-T6 was detected by thiazolyl blue assay under 24 h interventions with 0, 20, 50, 80 μmol/L AM-1241 and 0, 10, 20, 30, 40 μmol/L AM-630, respectively. Besides control group, the remaining groups were well cultured in low-glucose DMEM containing 100 mU/L glucose oxidase (GO) for 12 h to prepare the oxidative stress model. Then, AM-1241 intervention group was treated with 50 μmol/L low-glucose DMEM medium. After incubation for 12 h, the AM-1241+AM-630 antagonist group was treated with CB2 antagonist AM-630 (20 μmol/L) for 2 h, and cultured with 50 μmol/L AM-1241 in complete low-glucose medium for 12 h. The optimal drug concentration was selected according to the cell viability considered by the experiment results. Type III collagen (C III) content in the HSC-T6 supernatant was detected by enzyme-linked immunosorbent assay. Glutathione (GSH) content in HSC-T6 was detected by spectrophotometry. CB2 and heme oxygenase-1(HO-1) in each group of HSC-T6 were detected by western blotting.@*Results@#HSC-T6 proliferation was inhibited in each group of AM-1241 in a concentration-dependent manner (P < 0.05). The inhibition was highest at 80μmol/L, and the cell survival rate was (41.61% ± 3.13%) (P < 0.05). AM-630 concentration group had no significant inhibitory effect on the proliferation of HSC-T6 (P > 0.05). HSC-T6 expressed CB2 receptor in each group. The expression level of CB2 in the AM-1241 intervention group was higher compare with control group (P < 0.05).The expression of Col III were significantly higher in oxidative stress group (P < 0.05) than in control group, and the expression of Col III of AM-1241 intervention group was significantly lower than that in oxidative stress group (P < 0.05). Col III level in AM-1241+AM-630 antagonistic group was significantly higher than that in AM-1241 intervention group (P < 0.05). There was no significant difference between AM-1241+AM-630 antagonistic group and oxidative stress group (P > 0.05). The content of GSH and HO-1 in oxidative stress group was higher (P < 0.05) than control group. The content of GSH and HO-1 in the AM-1241 intervention group was higher compared with oxidative stress group, while content of AM-1241 + AM-630 antagonist group was lower compared to AM-1241 intervention group (P < 0.05), and the differences were not statistically significant for oxidative stress group.@*Conclusion@#CB2 agonist AM-1241 can inhibit the proliferation and activation of HSC-T6 and its mechanism may activate the nuclear translocation of Nrf2 binding to HSC-T6, initiating the up-regulation of antioxidant enzymes HO-1 and GSH protein expression, and thus increase the antioxidant effect of HSC-T6.

Exploration of enzyme-MnO2 nanosheets hybridization probe for sensitively colorimetric self-indicating of glucose / 中国药科大学学报

A colorimetric self-indicating probe for glucose was constructed by self-assembly of MnO2 nanosheets (MnO2 NSs) and glucose oxidase(GOD) in this paper.Under the weak acidic medium,glucose oxidase specifically catalyzes glucose into gluconic acid and hydrogen peroxide.The by-product of hydrogen peroxide could efficiently dissolve the MnO2 nanosheets,resulting into a significant decrease of the characteristic absorbance at 374 nm assigned to MnO2 NSs.Furthermore,the absorbance difference was linearly proportional to the concentration of glucose ranging from 1 to 20 μmol/L The fitted curve could be used for quantification of glucose with a correlation coefficient of 0.990 1.And the detection limit as low as 0.1 μmol/L could be reached based on the definition of three times of the deviation of the blank signal (3σ) and there was negligible interference with other co-existing amino acids,anions,cations and protein,which indicated high sensitivity and selectivity of the hybrid probe.The construction strategy of designated probe is readily generalized in principle for detection of numerous analytes in view of reactive property of MnO2 and the diversity of enzymes.

Preparation of Graphene/Gold Nanoparticles/Glucose Oxidase Flexible Electrode Based on Poly(ethylene terephthalate) Film and Its Application in Detection of Glucose / 分析化学

The graphene prepared by chemical vapor deposition onto copper foils was transferred onto a poly(ethylene terephthalate) (PET) soft substrate by the aid of poly-methyl methacrylate (PMMA).The soft G/AuNPs/GOD composite electrode based on PET substrate was fabricated using a protocol in which the uniform distribution of Au nanoparticles (AuNPs) was firstly obtained by controlling the evaporation of gold sol on a graphene surface, then thioglycolic acid (TGA) was modified on the AuNPs through Au-S bond, and finally glucose oxidase (GOD) was immobilized on the surface of AuNPs through acylation reaction between TGA and the GOD.Glucose was detected in the linear range from 0.05 to 10.55 mmol/L with a linear correlation coefficient (r) of 0.9955.The detection was performed in phosphate buffer solution (pH 7) at 25℃ with a working potential of 0.6 V (vs.SCE electrode), and the detection limit was 1 μmol/L (3σ).The G/AuNPs/GOD flexible electrode based on PET substrate provided a new pathway to detect glucose in special environments using wearable equipment, which enlarged the applied field of glucose detection.

Analysis on interference effects of vitamin C on three kinds of glucose detection methods / 国际检验医学杂志

Objective To investigate the interference effects of vitamin C on three different detection methods of blood glucose.Methods According to CLSI EP7-A2 document,5% volume of vitamin C(series concentrations) solution was added to fresh mixed serum,then hexokinase method(A),glucose oxidase method(B) and glucose reductase electrode method(C) were used to detect the level of fresh mixed serum glucose.The interference level of vitamin C on three different detection methods of blood glucose was evaluated.Results In 1 250 mg/L vitamin C,there was no interference on the method A for detecting blood glucose.In 156 mg/L vitamin C,it caused unacceptable negative interference on the method B and unacceptable positive interference on the method C.Moreover the interference degree of these two methods was increased with vitamin C concentration increase,but the same concentration of vitamin C had no significant difference on detecting different blood glucose levels by the same detection method.Conclusion High concentration of vitamin C can cause significant interference on glucose detection by glucose oxidase method and glucose reductase electrode method.

Structural regulation by calcium ion in preparing cross-linked enzyme aggregates / 生物工程学报

We studied the effect of calcium ion on particle size and pore structure of cross-linked enzyme aggregates (CLEAs) of glucose oxidase, with activity and stability of the enzyme as evaluation criteria. With calcium ion to prepare CLEA significantly decreased particle sizes of CLEAs whilst the pore structures of CLEAs gradually disappeared with the increase of calcium concentration. When glucose oxidase was precipitated at 0.1 mmol/L Ca²⁺, glucose oxidase in CLEA showed the definitive pore structure. Moreover, glucose oxidase activity in CLEA with Ca²⁺ was 1.69 times higher than that without Ca²⁺. Even at Ca²⁺ as high as 1.0 mmol/L, glucose oxidase activity in CLEA was 42% higher than that of CLEA without Ca²⁺. Furthermore, CLEA prepared with 0.1 mmol/L Ca²⁺ not only exhibited higher substrate conversion and operational stability, but also increased the maximum reaction speed. Therefore, calcium ion improved the performance of glucose oxidase in CLEAs.

High-level production of glucose oxidase by recombinant Pichia pastoris using a combined strategy / 生物工程学报

To enhance the production of glucose oxidase by recombinant Pichia pastoris, two strategies were developed, which were namely co-feeding of methanol and sorbitol and co-expressing of the protein disulfide isomerase (PDI) and Vitreoscialla hemoglobin (VHb). The volumetric activity reached 456 U/mL by using the strain X33/pPIC9k-GOD, in 5 liter fermentator, with the co-feeding of methanol and sorbitol, it was 0.2 fold higher than that only feeding by methanol. The improved strain was obtained by co-expressing PDI-VHb with GOD. While fermented in a 5 liter fermentator by feeding methanol and sorbitol, the activity of the improved strain reached 716 U/mL with a yield of 7 400 mg/L total soluble protein concentration. These results indicated that heterologous protein expression level can be enhanced by optimizing fermentation condition and co-expression molecular chaperon in Pichia pastoris.

Biosensors: A Sensitive Strategy to Detect Glucose in Body Fluids.

Over the past decades diabetes is one of the leading causes of mortality and morbidity in the world, thus inventing glucose biosensors with accurate continuous monitoring is of growing concern amongst the scientists worldwide. This manuscript reviews the development of glucose biosensors over the last 50 years since the invention of the first glucose sensing electrode and various approaches considered to develop accurate and modern techniques of glucose sensing. This review provides brief introduction to principles of various glucose biosensors with systemization and classification of glucose monitoring principles. Thus the main aim of this manuscript is to check history of glucose biosensors, comment on their current status and commercial aspects, and examine future challenges.

Performance Evaluation of BAROZEN H, a Networking Blood Glucose Monitoring System for Medical Institutions

BACKGROUND: We evaluated the analytical performance of Barozen H (i-SENS Inc., Korea), a new glucometer equipped with networking function for medical institutions, according to the ISO 15197:2003 and ISO/DIS 15197:2011 guidelines. METHODS: We measured the precision of 10 Barozen H glucometers, in terms of repeatability and intermediate precision, and determined their accuracy relative to that of automatic chemistry analyzer AU5421 (Beckman Coulter, USA). Three other glucometers-Precision PCx (Abbott, USA), Glucocard Sigma (Arkray, Japan), and SureStep Flexx (Johnson & Johnson, USA) were also evaluated, and their accuracies and hematocrit interferences were compared. RESULTS: The standard deviation and coefficient of variation of Barozen H for repeatability and intermediate precision were 0.11-0.15 mmol/L and 2.3-3.6%, respectively. With respect to accuracy, in accordance with ISO 15197:2003 criteria, Barozen H yielded 98.0% of results within +/-0.83 mmol/L or +/-20%. Further, per the ISO/DIS 15197:2011 criteria, 95.2% of results were within +/-0.83 mmol/L or +/-15%; Barozen H was the only glucometer satisfying the more stringent ISO/DIS 15197:2011 criteria. Error grid analysis showed that all results from Barozen H were in zone A, indicating its excellent clinical accuracy. Hematocrit, ranging from 20% to 60% did not cause any significant interference. CONCLUSIONS: Barozen H showed excellent analytical performance, and it was the most clinically accurate glucometer tested. It can be expected to provide reliable results satisfying ISO/DIS 15197:2011 as well as ISO 15197:2003 criteria.

Performance Evaluation of BAROZEN H, a Networking Blood Glucose Monitoring System for Medical Institutions

BACKGROUND: We evaluated the analytical performance of Barozen H (i-SENS Inc., Korea), a new glucometer equipped with networking function for medical institutions, according to the ISO 15197:2003 and ISO/DIS 15197:2011 guidelines. METHODS: We measured the precision of 10 Barozen H glucometers, in terms of repeatability and intermediate precision, and determined their accuracy relative to that of automatic chemistry analyzer AU5421 (Beckman Coulter, USA). Three other glucometers-Precision PCx (Abbott, USA), Glucocard Sigma (Arkray, Japan), and SureStep Flexx (Johnson & Johnson, USA) were also evaluated, and their accuracies and hematocrit interferences were compared. RESULTS: The standard deviation and coefficient of variation of Barozen H for repeatability and intermediate precision were 0.11-0.15 mmol/L and 2.3-3.6%, respectively. With respect to accuracy, in accordance with ISO 15197:2003 criteria, Barozen H yielded 98.0% of results within +/-0.83 mmol/L or +/-20%. Further, per the ISO/DIS 15197:2011 criteria, 95.2% of results were within +/-0.83 mmol/L or +/-15%; Barozen H was the only glucometer satisfying the more stringent ISO/DIS 15197:2011 criteria. Error grid analysis showed that all results from Barozen H were in zone A, indicating its excellent clinical accuracy. Hematocrit, ranging from 20% to 60% did not cause any significant interference. CONCLUSIONS: Barozen H showed excellent analytical performance, and it was the most clinically accurate glucometer tested. It can be expected to provide reliable results satisfying ISO/DIS 15197:2011 as well as ISO 15197:2003 criteria.

Isolation and Identification of Glucose Oxidase Hyper Producing Strain of Aspergillus niger.

Aims: Glucose oxidase is an enzyme with large scale applications in various industries. It is also used in several diagnostic kits which makes it medically important as well. Our aim was to isolate indigenous glucose oxidase hyper producing strain of Aspergillus niger from different soil samples of Punjab, Pakistan. Study Design: An experimental study. Place and Duration of Study: Institute of Industrial Biotechnology, GC University, Lahore from March 2011 to July 2012. Methodology: Two hundred and seventy nine fungal strains were isolated from soil of different localities of Punjab. Isolates were screened for glucose oxidase production using submerged fermentation. Glucose oxidase hyper producer isolate was identified using morphological and molecular techniques i.e. 18S rDNA. DNA was isolated and amplified using PCR. Gene sequencing was done and homology analysis was studied. Rate of glucose oxidase production was also analysed. Results: Glucose oxidase hyper producing isolate was identified as A. niger A247 strain. This strain gave best reproducible results (145.22 ±0.034 U/g of cell mass) after 72 hrs of fermentation at 30ºC and at a medium pH of 7.2. Conclusion: Our results indicate the natural ability of A. niger to produce Glucose oxidase in large quantity instead of using genetic manipulation techniques.

Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%), DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, respectively, which after desalting was 15.52 and 12.05 U mg-1, and after ion exchange and gel filtration chromatography was 29.09 - 62 and 25.72 - 59.37 U mg-1 for A. niger and P. notatum, respectively. The antimicrobial activity was determined by disc diffusion method against selected microbial strains where glucose oxidase from A. niger showed anti-bacterial activity, while no fungicidal effects were shown by both A. niger and P. notatum glucose oxidases.

Production of rabbit antibodies against purified Glucose oxidase

Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.