Dissertations / Theses on the topic 'Glucose oxidase/glucose reaction'

This work describes the integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reactor for selective determination of the enzyme substrate. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre- or post- separation chemical modification of the analyte. The overall goal of this work is to develop a system in which sample introduction, separation of the analyte/substrate from other biological species, enzymatic conversion of the analyte/substrate into a detectable product, and sensitive detection are all included within a single analysis scheme. Immobilization of the enzyme is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the immobilized glucose oxidase produces H2O2 which migrates the length of the capillary under the influence of electroosmotic flow and is detected amperometrically at the capillary outlet. The optimal response, kinetics, and stability for the enzyme reactor are determined through characterization of several parameters including the concentration ratio of PSS:GOx, applied separation voltage, and the inner diameter of the separation capillary. Various analyte mixtures containing the substrate and other biological species were evaluated to illustrate selective separation and determination of the substrate from other biomolecules. Optimization of this electrostatically assembled capillary enzyme reactor lead to application of these parameters to similar enzymes such as glutamate oxidase. Future application to similar enzymes like L-amino acid oxidase and possible microfluidic systems is a long-term goal of the system.
Ph. D.

A library of graphene-based hybrid materials was synthesized as novel hybrid electrochemical electrodes for electrochemical energy conversion and storage devices and electrocatalytical sensing namely enzymeless glucose sensing. The materials used were supercapacitive graphene-family nanomaterials (multilayer graphene-MLG; graphene oxide-GO, chemically reduced GO-rGO and electrochemical reduced GOErGO) and pseudocapacitive nanostructured transition metal oxides including cobalt oxide polymorphs (CoO and Co3O4) and cobalt nanoparticles (CoNP). These were combined through physisorption, electrodeposition, and hydrothermal syntheses approaches. This project was carried out to enhance electrochemical performance and to develop electrocatalytic platforms by tailoring structural properties and desired interfaces. Particularly, electrodeposition and hydrothermal synthesis facilitate chemically-bridged (covalently- and electrostatically- anchored) interfaces and molecular anchoring of the constituents with tunable properties, allowing faster ion transport and increased accessible surface area for ion adsorption. The surface morphology, structure, crystallinity, and lattice vibrations of the hybrid materials were assessed using electron microscopy (scanning and transmission) combined with energy dispersive spectroscopy and selected-area electron diffraction, X-ray diffraction, and micro-Raman Spectroscopy. The electrochemical properties of these electrodes were evaluated in terms of supercapacitor cathodes and enzymeless glucose sensing platforms in various operating modes. They include cyclic voltammetry (CV), ac electrochemical impedance spectroscopy, charging-discharging, and scanning electrochemical microscopy (SECM). These hybrid samples showed heterogeneous transport behavior determining diffusion coefficient (4⨯10-8 – 6⨯10-6 m2/s) following an increasing order of CoO/MLG < Co3O4/MLG < Co3O4/rGOHT < CoO/ErGO < CoNP/MLG and delivering the maximum specific capacitance 450 F/g for CoO/ErGO and Co3O4/ rGOHT. In agreement with CV properties, these electrodes showed the highest values of low-frequency capacitance and lowest charge-discharge response (0.38 s – 4 s), which were determined from impedance spectroscopy. Additionally, through circuit simulation of experimental impedance data, RC circuit elements were derived. SECM served to investigate electrode/electrolyte interfaces occurring at the solid/liquid interface operating in feedback probe approach and imaging modes while monitoring and mapping the redox probe (re)activity behavior. As expected, the hybrids showed an improved electroactivity as compared to the cobalt oxides by themselves, highlighting the importance of the graphene support. These improvements are facilitated through molecular/chemical bridges obtained by electrodeposition as compared with the physical deposition.

A sacarose é uma matéria-prima em franca expansão de produção no Brasil, seu maior produtor e exportador. Essa molécula pode ser convertida, através de um processo multienzimático, em produtos de maior valor agregado: frutose e ácido glicônico, os quais são importados pelo país, e amplamente utilizados em indústrias químicas, de produção de fármacos e setores alimentícios. Neste estudo, avaliou-se a hidrólise da sacarose pela invertase assim como a conversão da glicose em ácido glicônico, pela ação da glicose oxidase, ambas em processo descontínuo-alimentado. A solução de substrato (64g/L-sacarose; 32g/L-glicose) foi adicionada segundo as seguintes leis: constante, linear crescente, linear decrescente, exponencial crescente e exponencial decrescente. No caso da glicose, foi necessária a utilização de enzima auxiliar, a catalase, para degradar a água oxigenada formada durante a conversão da glicose. Mediante os resultados dos testes com os dois substratos, realizou-se teste de conversão direta da sacarose em frutose e ácido glicônico, utilizando-se invertase, glicose oxidase e catalase em regime descontínuo-alimentado, com alimentação linear decrescente (melhor resultado para ambos os substratos). No procedimento contínuo, alvo principal do trabalho, utilizou-se reator com membrana, da marca MILLIPORE ®, integrando em uma única etapa a conversão catalítica, a separação/concentração do produto e a recuperação do biocatalisador. A temperatura foi controlada por circulação de água, tendo acoplado uma bomba peristáltica (para controlar a vazão de alimentação do substrato) e um sistema de pressurização. O reator operou com membrana de ultrafiltração (corte molecular = 100 kDa) e foi mantido sob agitação constante. Os parâmetros de partida foram, a princípio, fixados de acordo com os valores otimizados no reator descontínuo-alimentado com o emprego simultâneo das enzimas.
Sucrose is a commodity largely produced in Brazil and one of the most used and commercialized product in food industry. It can be converted through a multienzyme process in fructose and gluconic acid, which have commercial values higher than sucrose. Both products are imported by Brazil, being largely employed in the chemical, food and pharmaceutical industry. This work dealt with the hydrolysis of sucrose by invertase into fructose and glucose, and the oxidation of glucose to gluconic acid by glucose oxidase and catalase. Catalase was added in order to decompose the hydrogen peroxide an inhibitor of glucose oxidase formed as by-product of the oxidation. Two processes were employed. Fed-batch in which the hydrolysis and oxidation reactions were carried out separately by adding invertase followed by glucose oxidase and catalase was conducted by adding the solution of substrate according to a constant, increasing linear, decreasing linear, increasing exponential or decreasing exponential mode. The best fed-batch performance was attained through the decreasing linear addition of sucrose (64g/L) and glucose (32g/L). Setting this kind of addition and using all enzymes simultaneously, the direct conversion of sucrose to fructose and gluconic acid occurred at a yield of 72%. The continuous process was carried out in a cell-type membrane reactor (membrane cut off = 100 kDa), in which the sucrose conversion was made by using all enzymes simultaneously, leading to a final yield of about 76%

De nombreuses réactions enzymatiques sont à l’origine de processus physiologiques au sein des organismes vivants. Ces réactions sont basées sur des transferts de protons et d’électrons et con-duisent souvent à la production d’espèces secondaires. Parmi elles, les espèces réactives de l’oxygène et de l’azote (ROS, RNS) présentent un intérêt particulier puisqu’elles jouent un double rôle : d’une part en permettant à l’organisme de réagir à un stress par l’activation de voie de signalisation redox, et d’autre part ces ROS et RNS peuvent causer des dommages tissulaires et être à l’origine de dys-fonctionnement (stress oxydant) au sein de l’organisme. La haute réactivité de ces espèces induit leurs faibles durées de vie (ns-min) et rend l’étude de certaines réactions enzymatiques difficiles en solu-tion. Ce projet de thèse a pour objectif de développer un microréacteur biomimétique pour l’étude d’activités enzymatiques produisant des ROS/RNS. En effet, en confinant une réaction au sein d’un compartiment de taille équivalente à celle d’une cellule (20-100 μm de diamètre), les espèces générées (H2O2, NO•, NO2-) doivent pouvoir être sondées in situ avec une résolution cinétique et quantitative. Des vésicules unilamellaires géantes sont formées en conditions physiologiques et servent de micro-réacteurs pour l’analyse des activités enzymatiques de la glucose oxydase et des NO-synthases. La microscopie de fluorescence permet l’observation des vésicules et le suivi du déclenchement de la réaction assuré par microinjection. Les espèces produites sont ensuite détectées en temps réel par électrochimie afin de déchiffrer à terme les différentes voies enzymatiques des NO-Synthases
Enzymatic reactions are involved in many physiological phenomena in living organisms. These reactions are based on protons and electrons transfers and can lead to the production of by-products. Among them, reactive oxygen and nitrogen species (ROS and RNS) are of great interest as they play a double role: on the one hand by allowing the organism to react to a stress by the activation of signaling redox pathways, and on the other hand, ROS and RNS can cause oxidative damages to tissues ensuing dysfunctions in the organism. The high reactivity of such species induce their short lifetimes (ns-min) and leads to uncertainties when it comes to the study of some enzymatic reactions in bulk. This PhD project aims to develop a biomimetic microreactor for the study of enzymatic ac-tivities producing ROS/RNS. Indeed, by confining a reaction within a cell-sized compartment (20-100 μm diameter), the generated species (H2O2, NO•, NO2-) could be analyzed in situ with a quantita-tive and kinetic resolution. Giant unilamellar vesicles are formed in physiological conditions and are used as microreactors for the monitoring of enzymatic activities of glucose oxidase and NO-synthases. Fluorescence microscopy allows individual vesicle observation and the monitoring of reactions trig-gered by microinjection. Then, released species are detected in real-time by electrochemistry in order to decipher the diverse enzymatic pathways of NO-Synthases

Amyloid fibrils are a misfolded state formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them an excellent target for enzyme immobilisation and their strength, stability and nanometre size are attractive features for exploitation in the creation of new bionanomaterials. The aim of this thesis was to functionalise amyloid fibrils by conjugation to glucose oxidase (GOD). GOD is a relatively stable glycoprotein that catalyses the oxidation of glucose and the release of hydrogen peroxide. The consumption of glucose can be measured to assess glucose levels, and the release of hydrogen peroxide is cytotoxic to cells and is thus an effective antibacterial agent. Three methods of attachment were used: cross-linking using glutaraldehyde, periodate oxidation of the glycoprotein shell, and cross-linking using glutaraldehyde following deglycosylation. GOD retained activity upon attachment by all three methods. These attachment methods were assessed using electrophoresis, centrifugation, sucrose gradient centrifugation and TEM. Gel electrophoresis indicated a high degree of cross-linking and TEM showed no significant change of fibril morphology upon cross-linking. Centrifugation experiments suggested a non-covalent interaction was occurring between amyloid fibrils and GOD, and a covalent attachment was occurring upon addition of glutaraldehyde. Sucrose gradient centrifugation provided increased separation of cross-linked material compared to other separation methods, and showed greater cross-linking to crystallin amyloid fibrils than insulin fibrils. Cross-linking native GOD using glutaraldehyde was chosen for further experiments, as it was found to be most effective for GOD attachment to amyloid fibrils. The resulting functionalised enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The distribution of the functionalised fibrils through the film was characterised using SEM and confocal microscopy, where film components were found to be unevenly dispersed. The antibacterial effect of the functionalised film was then tested on E. coli and the antifungal effect of the film was tested on Fusarium, Rhizopus and Penicillium. Growth of E. coli was inhibited around functionalised film circles, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. However, no growth inhibition of fungal species was observed. This work is of significance as it demonstrates the ability to convert a waste material, bovine lens crystallin, to high value protein nanofibres and incorporate functionality via GOD attachment. The incorporation of the GOD-functionalised amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalised amyloid fibril scaffold. Future development of this model system has the potential to lead to the production of a novel biomaterial for use in food packaging due to the antimicrobial properties of GOD.

Frutose e Ácido Glicônico são produtos importados empregados em diferentes setores nas áreas química, farmacêutica e alimentícia, representando um mercado de dois milhões de dólares (US$ 2,0 milhões) por ano. Por sua vez, a sacarose pode ser empregada como matéria-prima para a obtenção destes produtos através de conversão enzimátiva empregando invertase e glicose-oxidase. O uso de biorreatores com membrana (MBR) mostra-se interessante em processos enzimáticos, pois, ao serem empregados em processos contínuos permitem, simultaneamente, produção e separação dos produtos, reduzindo a formação de subprodutos e, eventual, inibição da enzima por excesso de substrato ou produtos. A sacarose é convertida em xarope de açúcar invertido (solução equimolar de frutose e glicose) pela invertase (Bioinvert®, enzima comercial), seguido pela oxidação da glicose em ácido glicônico pela ação da glicose oxidase (GO). O processo de conversão multi-enzimático da sacarose foi obtido através da alimentação de sacarose (50 mM) em reator com membrana (MBR) contendo invertase (24 U/mL), glicose-oxidase (0,5 U/mL) e catalase (470 U/mL) e operando com vazão específica de 6,0 h-1, 35ºC e pH 5,5. As condições operacionais otimizadas possibilitaram a conversão completa da sacarose (X = 100 %) e da glicose resultante (Y = 100%) com velocidades específicas de reação de 4,2 mmol/U.h, 0,60 mmol/U.h e 0,00062 mmol/U.h, respectivamente, para a invertase, glicose oxidase e catalase. A respeito da oxidação da glicose, a adição de catalase no meio reacional se fez necessária para minimizar os efeitos inibitórios sobre a GO através do peróxido de hidrogênio formado.
The fructose and gluconic acid are products of great application in chemical, pharmaceutical and food industry. The actual Brazilian market for these compounds is about US$ 2 millions, here as the sucrose, the raw-material used for their production, represents about 2.4% of the Brazil\'s GNP. This conversion increases the value added to the sugarcane, usually marketed as a commodity, because the fructose and gluconic acid are more valuable products than sucrose. The use of membrane bioreactor (MBR), which operates under mild conditions regarding internal pressure, temperature and pH, has been growing along the years for enzyme catalyzed processes. Moreover, in the MBR the reaction and separation of the products occur simultaneously, avoiding the formation of by-products and the eventual inhibition of the enzyme caused by excess of substrate or products. The sucrose is converted to the inverted syrup (an equimolar solution of fructose and glucose) by invertase (in this work was employed Bioinvert®, a commercial invertase) followed by the oxidation of glucose in gluconic acid by the glucose oxidase (GO). The multi-enzymatic conversion of sucrose was attained when carried out under initial substrate of 50mM and invertase, glucose oxidase and catalase concentrations, respectively, of 24.0 U/mL, 0.5 U/mL and 470 U/mL in a membrane reactor utilizing a dilution rate of 6.0 h-1, 35ºC and pH 5.5. The optimized operational conditions led to a conversion yield of 100% for sucrose hydrolysis and glucose oxidation steps resulting in enzyme productivity of 4.2 mmol/U.h, 0.60 mmol/U.h and 0.00062 mmol/U.h, respectively, to invertase, glucose oxidase and catalase. In regard to the glucose oxidation, the addition of catalase in the reaction medium was necessary, in order to minimize the inhibition of the GO by the hydrogen peroxide formed.

The main goal of this research was to develop an easy to prepare and sensitive biosensor that would be able to detect glutamate in solution using ionic self-assembly methods. This was accomplished by preparing an ionically-self-assembled monolayer that included an electron transport mediator and an enzyme that would generate a current proportional to the concentration of analytes in solution. Biosensors were produced for the detection of glucose and glutamate. Ferrocene poly(allylamine) (FePAA) was assembled on negatively charged self-assembled monolayer and shown to be electrostatically bound by cyclic voltammetry. Model films were made of FePAA and poly(styrenesulfonate) to determine if multilayer films could be assembled using electrostatic assembly. These experiments demonstrated that 7 bilayers is the maximum number of bilayers oxidizable by the heterogeneous reaction at the electrode surface. ISAMs were then assembled on a 2 mm gold electrode and on a gold fiber microelectrode using FePAA and glucose oxidase. Using cyclic voltammetry, these ISAMs were shown to be able to oxidize glucose in solution. The LOD was determined to be lower for the microelectrode than for the 2 mm gold electrode, which was expected, while both compared well to the literature. The Km? were found to be smaller than other glucose biosensors while the Icat increased with increasing number of bilayers. This demonstrated that the GluOx is making good electrical contact with the layer below. These glucose oxidase ISAMs, however, do not exhibit structural stability in flow-injection experiments. As a solution to the ISAM modified electrodes degrading in the flowing system, a covalently modified surface was developed. Using cyclic voltammetry, these covalently modified surfaces were shown to be able to oxidize glucose in solution. The LOD of the covalently modified 2 mm gold electrode was calculated to be lower than the 2 mm ISAM modified gold electrode, due to the fast heterogeneous kinetics on the covalently modified electrode surface. The Km? and Icat for the covalently modified 2 mm gold electrode were found to be the similar to the 2 mm ISAM modified gold electrode indicating that the covalently modified electrodes will be a suitable replacement. The covalently modified surfaces exhibit excellent structural stability and detect much lower glucose amounts in flow-injection experiments. ISAMs were subsequently assembled on gold fiber microelectrodes using FePAA and glutamate oxidase. Glutamate was able to be detected in solution at biologically significantly quantities using cyclic voltammetry. The Km? was shown to be comparable to literature values and Icat was shown to increase with increasing number of bilayers. These results demonstrate that an ISAM constructed using FePAA/GlutOx is a feasible way to detect glutamate in a system.
Ph. D.

Thesis (MSc)--Stellenbosch University, 2002.
Full text to be digitised and attached to bibliographic record.
ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has established its importance as a wine yeast and also proven itself as a reliable starter culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly, its metabolic activities result in the production of higher alcohols, fatty acids and esters, which are important flavour and aroma compounds that are essential for consistent and predictable wine quality. There is a growing consumer demand for wine containing lower levels of alcohol and chemical preservatives. Glucose oxidase (GOX) has received considerable research interest regarding its potential application in the wine industry to reduce alcohol levels and as a biocontrol agent. Several physical processes are used for the removal or reduction of alcohol in wine and some of them are sometimes used in combination. These processes tend to involve expensive equipment and can be intensive from a processing point of view. An alternative approach was introduced with the concept of treating grape must with GOX to reduce the glucose content of the must, and therefore produce a wine with a reduced alcohol content after fermentation. Due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialised wine yeast strains possessing a wide range of optimised, improved or novel oenological properties. The first and main objective of this study was to genetically engineer wine yeasts to produce wine with a reduced alcohol content. In order to do this, the structural glucose oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5) containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox- PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of S. cerevisiae (~1278). Results obtained indicated the production of biologically active glucose oxidase and showed that it is secreted into the culture medium. This would mean that the enzyme will convert the glucose to gluconic acid in the medium before the yeast cells are able to metabolise the glucose to ethanol. Microvinifications performed with Chardonnay grapes showed that the laboratory yeast starter cultures transformed with GOX1 were indeed able to reduce the total amount of alcohol in the finished product. The second objective of this study involved the potential application of GOX as a biocontrol agent. Screening was performed for wine spoilage microorganisms, such as acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage microorganisms tested formed different sized inhibition zones, indicating varying degrees of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed under a scanning electron microscope. The total collapse of the bacterial cell wáll could be seen and might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a result of the peroxidation of the membrane lipid, and a strong oxidising effect on the bacterial cell, which is the cause of the destruction of basic molecular structures, such as nucleic acids and cell proteins. In this exciting age of molecular yeast genetics and modern biotechnology, this study could pave the way for the development of wine yeast starter culture strains for the production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs) within the wine industry is a limiting factor at present and credible means must be found to effectively address the concerns of traditionalists within the wine industry and the negative overreaction by some consumer groups. There is a vast potential benefit to the wine consumer and industry alike and the first recombinant wine products therefore should unmistakably demonstrate safe products free of potentially harmful compounds, and have organoleptic, hygienic and economic advantages for both the wine producer and consumer.
AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na wyne met 'n laer alkoholinhoud en minder preserveermiddels. Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en selfs unieke, wynkundige einskappe bevat. Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278) getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei. Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied (H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese molekulêre strukture, soos die nukleïensure en selproteïene. Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste, asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan. Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese voordele toon te opsigte van beide die wynprodusent en gebruiker.

A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.

Electrochemical polymerization of pyrrole functionalized polystyrene (PStPy) with pyrrole was carried out in water-sodium dodecyl sulfate solvent-electrolyte couple. Characterization of the resulting copolymer was performed via Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and four probe conductivity measurements. Glucose oxidase and polyphenol oxidase enzymes were immobilized in polypyrrole (PPy) and conducting copolymer of pyrrole functionalized polystyrene with pyrrole (P(PStPy-co-Py). Resulting enzyme electrodes were characterized by kinetic parameters
Vmax and Km. Behavior of enzyme electrodes upon temperature and pH changes were investigated. Glucose oxidase electrode was used for the determination of glucose in orange juice and polyphenol oxidase electrode was used for the determination of polyphenolic compounds in red wine.

Insect herbivores often secrete glucose oxidase (GOX) onto plants to counteract plant defenses and potential pathogens. Whether generalist herbivores always have significantly higher GOX activities than their specialist counterparts at any comparable stage or conditions and how this is realized remain unknown. To address these two general questions, we subjected larvae of a pair of sister species differed mainly in host range, the generalist Helicoverpa armigera and its specialist counterpart Helicoverpa assulta, to the same sets of stage, protein to digestible carbohydrate (P:C) ratio, allelochemical or host plant treatments for simultaneous analyses of GOX transcripts and activities in their labial glands. GOX activity and transcripts are upregulated concurrently with food ingestion and body growth, downregulated with stopping ingestion and wandering for pupation in both species. The three tested host plants upregulated GOX transcripts, and to a lesser extent, GOX activity in both species. There were significant differences in both GOX transcripts and activity elicited by allelochemicals, but only in GOX transcripts by P:C ratios in both species. GOX activities were higher in H. armigera than H. assulta in all the comparable treatments, but GOX transcripts were significantly higher either in generalists or in specialists, depending on the developmental stages, host plants, P:C ratio and allelochemicals they encounter. These data indicate that the greater GOX activity in generalist herbivores is not achieved by greater transcription rate, but by greater transcript stability, greater translation rate, better enzyme stability and/or their combination.

Thesis (PhD)--Stellenbosch University, 2010.
ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized by intense fruitiness and varietal flavors. However, lengthy maturation of grapes in the vineyard does not only translate into higher flavor intensity but also into higher sugar levels, which, in turn, leads to wines with higher concentrations of alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced. This, along with health issues and anti-social behavior linked to high-risk alcohol consumption patterns, stricter legislation and increased tax rates associated with high-alcohol wines, have increased demand for wines with reduced alcohol concentrations, without loss of the intense fruity aromas. Although low-alcohol wines can be made using physical post-fermentation processes, such approaches are often expensive and can impact adversely on wine flavor. As an alternative strategy, yeast strains are being developed by several research groups to convert some of the grape sugars into metabolites other than ethanol. Based on promising results from previous preliminary work, this study focused on the development of an industrial Saccharomyces cerevisiae wine strain producing glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4). GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly, thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2 and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes were each integrated into the genome of two laboratory yeast strains (BY4742 and S1278b) and one industrial wine yeast strain (VIN13). An additional integration cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S. cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained by integrating a single copy of GOX2LOX into the IME1 locus. To generate an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation, micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine yeast transformants were sufficiently high to conduct follow-up small-scale wine fermentation trials. The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental winemaking conditions. Results from this work indicated that glucose oxidase was produced and secreted by VIN13-D1 that dominated the fermentation to the end, but also that the enzyme was not highly active under the evaluated winemaking conditions. Consequently, no significant decrease in ethanol concentrations was observed in the wine made from VIN13-D1 when compared to that from VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry (FT-MIR) to determine the chemical composition and Gas chromatography with a flame ionization detector (GC-FID) to evaluate the concentrations of aroma compounds. The levels of gluconic acid were determined by enzymatic assays. Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight significant differences between the wines made by VIN13 (wild-type) and VIN13- D1. Chemometric projections of the score plots for all results allowed insight into all significant variation up to three principal components (PCA) or PLS components, which showed very clearly that GA is a key factor in evaluating the effect of GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13- D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds yielded better results, with the GC-compounds displaying greater discriminability between cultivars and VIN13 / VIN13-D1. It can be concluded from these results that the greatest influence of VIN13-D1 produced wines could be observed in the aroma components, but, because there were also discriminability effects discernable in the FT-MIR-compounds, thus the flavor components were also affected. The activity of GOX in grape juice was further investigated in controlled small scale fermentations performed in a bio-reactor. It was confirmed that GOX is active under aerobic conditions, inactive under anaerobic conditions, and can be activated instantly when an anaerobic culture is switched to aerobic conditions (simulated micro-oxygenation). These fermentations showed that glucose oxidase is active in grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it was shown with the help of a simplified model, that under ideal conditions, GOX secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined concentration for the production of low alcohol wines. This work gives more insight into how to employ a GOX-producing wine yeast during winemaking and strongly suggests the use of micro-oxygenation to activate the enzyme in order to reduce available glucose, thereby diverting a portion of the sugar carbon away from ethanol production.
AFRIKAANSE OPSOMMING: Gewilde wynstyle word dikwels gemaak van volryp, goed ontwikkelde druiwe, gekarakteriseer deur intense aromas en smaakkomponente wat direk met spesifike kultivars geassosieer word. ’n Nadelige gevolg om druiwe te lank aan die wingerdstok te laat bly hang sodat meer intense geurkomponente kan ontwikkel, is die toename in suikerinhoud. Hierdie addisionele suiker lei tot wyne met hoër alkoholvlakke. Te hoë alkoholvlakke kan wyn ongebalanseerd laat voorkom en die smaak nadelig beïnvloed. Dit, tesame met gesondheidsredes en anti-sosiale gedrag wat gekoppel kan word aan die inname van te veel alkohol, strenger wetgewing aangaande dronkbestuur en die toename in belasting op wyne met ’n hoër alkoholinhoud, het aanleiding gegee tot ’n aanvraag vir wyn met ’n verlaagte alkoholinhoud, sonder dat aroma- en geurkomponente ingeboet word. Alhoewel daar sekere fisiese/gemeganiseerde prosesse beskikbaar is om die alkohol in wyn te verwyder of te verminder, is ’n nadeel dat hierdie prosesse baie duur en arbeidsintensief is, en dat dit deur sommige wynpuriste as te ingrypend in die ‘natuurlike’ proses van wynmaak beskou word. Sommige van hierdie alkoholverwyderingsprosesse kan ook die wyn se geur- en aromakomponente nadelig beïnvloed. As alternatief tot hierdie fisies-chemiese prosesse word wyngiste reg oor die wêreld deur verskillende navorsingsgroepe ontwikkel sodat van die druifsuikers nie na alkohol omgeskakel word nie, maar eerder ander metaboliete. Belowende navorsingsresultate in ’n voorafgaande studie het aanleiding gegee tot hierdie navorsingsprojek. In hierdie studie word daar klem gelê op die ontwikkeling, deur middel van genetiese manipulering, van ’n industriële wynras van Saccharomyces cerevisiae sodat dit in staat sal wees om glukose-oksidase (GOX; b-D-glukose:suurstof oksidoreduktase, EC 1.1.3.4) te produseer. GOX kan reeds b-D-glukose in die medium oksideer na glukoonsuur (GA), wat sodoende verhoed dat dit verder gemetaboliseer word via glukolise, en dit het tot gevolg dat ’n gedeelte van die beskikbare suiker nie omgeskakel word na alkohol nie. Die strukturele glukose-oksidase-geen (GOX) van die voedsel-gegradiëerde fungus, Aspergillus niger is gebruik tydens die konstruksie van drie kassette (GOX1, GOX2 en GOX2LOX). Binne hiedie geenkassette is A. niger se GOX-geen se transkripsieinisiëring en -terminering onafhanklik deur die fosfogliseraat-kinase-1-promotor (PGK1P) en termineerder (PGK1T ) bewerkstellig. Om uitskeiding van GOX uit die gis te bewerkstellig, is daar van die a-spesifieke gisferomoon-a-faktor (MFa1S) in GOX1 gebruik gemaak, en in GOX2, van A. niger se eie natuurlike sekresiesein. Hierdie geenkassette is binne-in die genoom van twee labaratoriumgisrasse van S. cerevisiae (BY4742 en S1278b) asook een industriële wyngisras (VIN13) geintegreer. ’n Addisionele integreringskasset (die sogenaamde GOX2LOX-kasset) is gemaak om die IME1-geen van S. cerevisiae te elimineer. Binne die GOX2LOXkasset is GOX2 aan ’n loxP-kasset gekoppel. Die nuwe wyngis VIN13-D1 is verkry deur ’n genomiese integrasie van GOX2LOX binne-in die IME1-lokus. Om die niesporulerende GOX-produserende wyngis VIN13-D2 te verkry, is VIN13-D1 gesporuleer, onderwerp aan mikromanipulasie en toegelaat om te herdiploidiseer. Ontledings het aangedui dat (i) GOX2 aanleiding gegee het tot hoër vlakke van ekstrasellulêre glukose-oksidase aktiwiteit in vergelyking met GOX1; en (ii) dat die vlakke van uitgeskeide biologies-aktiewe glukose-oksidase vir die wyngisrasse aansienlik hoër was. Dit het verdere kleinskaalse wynfermentasies geregverdig. Die getransformeerde wyngis VIN13-D1 is op eksperimentele skaal in die maak van rooi- en witwyn geëvalueer. Ontledings van hierdie eksperimentele wyne het daarop gedui dat glukose-oksidase deur die VIN13-D1-gisselle geproduseer en suksesvol uitgeskei tydens die wynmaakproses is, en dat VIN13-D1 die fermentasie gedomineer het en die alkoholiese gisting voltooi het. Resultate het egter ook aangedui dat die geproduseerde glukose-oksidase nie baie aktief was onder die wynmaaktoestande wat in hierdie eksperimentele wynmaakproses gegeld het nie, en gevolglik was daar nie ’n drastiese verlaging in die alkoholvlakke sigbaar toe VIN13-D1 se wyne met VIN13 se wyne vergelyk is nie. Wynmonsters is deur middel van Fourier-transformasie-mid-infrarooispektroskopie (FT-MIR) ontleed ten einde die chemiese samestelling te bepaal, en gaschromatografie-massaspektrometrie (GCMS) is aangewend om die wynaromakomponente te bepaal. Die vlakke van glukoonsuur is deur middel van ensiematiese reaksies bepaal. Multiveranderlike data-analise [hoofkomponentanalise (PCA) en parsiële kleinte kwadrate (PLS1) diskriminantanalise] is op die data van die verskeie analitiese tegnieke toegepas om onderliggende veskille tussen die wyne van VIN13 (wilde-tipe) en VIN13-D1 uit te wys. Chemometriese projeksies het aangetoon dat daar wel beduidende variasies sigbaar was tot en met drie hoofkomponente en/of PLS-komponente wat duidelik aandui dat glukoonsuur ’n sleutelfaktor was ten opsigte van die uitwerking wat GOX op VIN13-D1- fermentasies in vergelyking met VIN13-fermentasies. VIN13-D1 effek manifestasies is die beste waargeneem op grafieke wat PLS1-diskriminantanalise-data bevat. Verder het PLS1-diskriminantanalise ook aangetoon dat van die ‘groepe’ wat gebruik was tydens die analise, die FT-MIR-komponente en die GC-komponente beter resultate gelewer het. Die GC-komponente het hulle verder daartoe geleen om tussen die verskillende kultivars en VIN13/VIN13-D1-fermentasies te diskrimineer. Daar kan dus met sekerheid gesê word dat die grootste invloed in VIN13-D1 wyne sigbaar is in die aromakomponent, maar omdat daar wel ook variasies sigbaar was in die MIR-komponente, dat die smaakkomponente ook beïnvloed was. Die aktiwiteit van GOX in druiwesap is verder ondersoek deur gebruik te maak van kleinskaalse fermentasies in bioreaktors. Daar is bevestig dat die VIN13-D1- geproduseerde GOX biologies-aktief was tydens aerobiese kondisies, onaktief was tydens anaerobiese kondisies, en onmiddelik geaktiveer kon word wanneer ’n anaerobiese fermentasie aerobies gemaak word (gesimuleerde mikro-oksigenasie). Hierdie verskillende fermentasies dui daarop dat glukose-oksidase inderdaat aktief is in druiwesap, en dat suurstof ’n sleutelfaktor is tydens die aktivering van die ensiem. Met behulp van ’n vereenvoudigde model kon aangetoon word dat tydens ideale toestande dit wel moontlik is om die alkoholvlakke te verlaag na ’n voorafbepaalde konsentrasie vir die bereiding van lae-alkohol wyne. Hierdie studie verskaf verdere insig hoe om ’n GOX-produserende wyngis gedurende die wynmaakproses vir die verlaging van die alkoholvlakke te benut. Verder is dit duidelik dat suurstof van kardinale belang is vir die aktivering van die glukoseoksidase- ensiem en dat ’n tegniek soos mikro-oksigenasie ’n belangrike rol in hierdie verband tydens die wynmaakproses sou kon speel.

In this study, an improved potentiometric intracellular glucose biosensor was fabricated with immobilization of glucose oxidase on a ZnO nanoporous material. The ZnO nanoporous material with a wall thickness around 200 nm was grown on the tip of a borosilicate glass capillary and used as a selective intracellular glucose sensor for the measurement of glucose concentrations in human adipocytes and frog oocytes. The results showed a fast response within 4 s and a linear glucosedependent electrochemical response over a wide range of glucose concentration (500 nM-10 mM). The measurements of intracellular glucose concentrations with our biosensor were consistent with the values of intracellular glucose concentrations reported in the literature. The sensor also demonstrated its capability by detecting an increase in the intracellular glucose concentration induced by insulin. We found that the ZnO nanoporous material provides sensitivity as high as 1.8 times higher than that obtained using ZnO nanorods under the same conditions. Moreover, the fabrication method in our experiment is simple and the excellent performance of the developed nanosensor in sensitivity, stability, selectivity, reproducibility and anti-interference was achieved. All these advantageous features of this intracellular glucose biosensor based on functionalised ZnO nanoporous material compared to ZnO nanorods demonstrate a promising way of enhancing glucose biosensor performance to measure reliable intracellular glucose concentrations within single living cells.

Original Publication:Alimujiang Fulati, Syed M. Usman Ali, Muhammad H. Asif, Naveed Ul Hassan Alvi, Magnus Willander, Cecilia Brännmark, Peter Strålfors, Sara I. Börjesson and Fredrik Elinder, An intracellular glucose biosensor based on nanoflake ZnO, 2010, Sensors and actuators. B, Chemical, (150), 2, 673-680.http://dx.doi.org/10.1016/j.snb.2010.08.021Copyright: Elsevier Science B.V., Amsterdam.http://www.elsevier.com/

ABSTRACT IMMOBILIZATION OF INVERTASE, POLYPHENOL OXIDASE AND GLUCOSE OXIDASE IN CONDUCTING COPOLYMERS OF THIOPHENE-CAPPED POLYTETRAHYDROFURAN AND PYRROLE Bö
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kbayram, AySe Elif Ph.D., Department of Chemistry Supervisor: Prof. Dr. Levent Toppare January 2005, 123 pages Immobilization of invertase, polyphenol oxidase (PPO) and glucose oxidase (GOD) enzymes were performed in electrochemically synthesized two types of conducting copolymers. One end and two end thiophene-capped polytetrahydrofuran (TPTHF-1 and TPTHF-2) were copolymerized with pyrrole under conditions of constant potential electrolysis. The copolymers were characterized by thermal, spectroscopic and scanning electron microscopy analyses. Immobilization was carried out via entrapment of enzymes in two types of matrices during the copolymerization of pyrrole with the insulating polymers in the presence of sodium dodecyl sulphate (SDS). Kinetic parameters: Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes. Temperature optimization, pH optimization, operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase and glucose oxidase were used to determine the amount of their substrates in samples. Polyphenol oxidase converts mono and diphenols to quinone. Amount of phenolic compounds in two kinds of wines were determined by analyzing the quinone amount. Glucose oxidase converts &
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-D-glucose to D-glucono-1,5-lactone. Glucose amount was determined in two kind of factory-produced orange juices by analyzing D-glucono-1,5-lactone.

Protein-based therapeutics have outstanding potential for the treatment of diseases, yet their physical and ' chemical protein instability has remained a major barrier for their rapid commercialisation. Development of efficient stabilisation protocols would be achieved by having a better understanding of the effect of key formulation parameters on protein stability. The main body of this project has been dedicated to investigating the effect of key formulation parameters, such as pH, ionic strength and temperature, on the stability and the aggregation mechanism of a model protein, Glucose oxidase at low (1 mg/ml) protein concentration. In addition, due to a strong trend in the pharmaceutical industry to formulate protein therapeutics at higher concentrations, key experiments were carried out at high (100 mg/ml) protein concentration. Changes in the physical state of the model protein were monitored using a combination of different techniques, including activity assay, Size-exclusion chromatography, visual assessment, intrinsic and extrinsic fluorescence spectroscopy, and static light scattering. In addition, we investigated whether the concept of accelerated stability study, widely used in the pharmaceutical industry, is a valid approach for predicting long-term stability. The results revealed that the rate and the mechanism of inactivation is pH, ionic strength, temperature, surfactant and protein concentration dependent. Furthermore, the results of the high concentration experiments showed the existence of an aggregation state, which appears to be different from the one observed at low protein concentrations. This is evident from the observation that · proteins at very high concentrations respond to formulations in a very different way than the same proteins at low concentrations. The findings from the work obtained from highly concentrated formulations showed the need for development of techniques that directly measure the stability of proteins at high concentrations without a dilution step. It also became evident that the concept of accelerated stability is not always applicable.

The aim of this project is to establish and understand the production of glucose oxidase and gluconic acid using the Aspergillus niger bioprocess and predict its response to operating conditions. Glucose oxidase and gluconic acid are produced by a wide range of microbes and have a variety of applications. In this study Aspergillus niger was chosen as the microorganism as it has the "generally accepted as safe" (gras) status in the U.S.A. It is also the major industrial producer. Glucose oxidase catalyses the conversion of glucose, oxygen and water to hydrogen peroxide and gluconic acid. This enzyme is used as a glucose and oxygen scavenger in the food industry and as a diagnostic tool in medicine for glucose determination. Gluconic acid is an organic acid used as a sequestering agent with a broad spectrum of applications. The world market for gluconic acid and its various salts was 45 000 metric tonnes in 1985 (Bigelis cited by Markwell et al. 1989). Gluconic acid and its derivatives can be produced using three technologies: electrolysis, mild chemical oxidation and bioprocess. The first two technologies have not been proven to be comnercially viable. The bioprocess offers diversity of feed and produces other products such as glucose oxidase. Literature has shown that the production of gluconic acid involves two kinetic areas. Firstly, the glucose oxidase enzyme must be induced. Secondly, glucose is converted to gluconic acid by the enzyme glucose oxidase. The factors affecting the kinetics associated with the induction of glucose oxidase have only been described qualitatively. Glucose, oxygen and pH have been shown to affect the induction of glucose oxidase. The effect of pH has been studied by Roukas and Harvey (1989) who found that induction, is maximal at a pH of between 5 and 6. The effect of glucose and oxygen have not been quantified. The kinetics of glucose oxidase conversion of glucose to gluconic acid have been well described by Atkinson and Lester (1974).

Ce memoire porte sur l'utilisation de solutions concentrees de nafion (gels hydrophobes de nafion) pour l'immobilisation de la glucose oxydase (god) a la surface d'electrodes. Dans un premier temps, l'oxydase est contenue au sein d'une pate de carbone aqueuse, le gel du ionomere realisant un interface selectif entre systeme enzymatique immobilise et milieu de dosage. Cette separation des deux intervenants est imposee par leur incompatibilite, la confrontation de la god et de la solution hydrophobe du polymere conduisant a une perte des proprietes catalytiques de l'enzyme. L'etude du comportement de ce dispositif permet d'en degager les caracteristiques et de mettre en evidences ses principales limitations: mauvaise immobilisation du mediateur (espece electroactive chargee du transport d'electrons entre enzyme et electrode), inhibition de la reaction d'oxydation du glucose, miniaturisation poussee impossible. Ce travail est complete par une etude cinetique, realisee au moyen d'une electrode tournante, grace a laquelle la permeation du glucose au sein des gels peut etre quantifiee. La plupart des problemes de la precedente electrode etant imputables a son organisation en couches successives, nous cherchons dans un deuxieme temps a associer de facon plus intime god et solution hydrophobe du polymere. L'inclusion de l'enzyme dans des gels dont les solvants sont des alcools d'assez faible poids moleculaire, puis dans des gels prealablement hydrates, donne des gels ou le pouvoir catalytique de la god est conserve. Ces materiaux enzymatiques sont aisement deposes a la surface d'electrodes, realisant des dispositifs dont les dimensions peuvent etre reduites et dont la structure permet d'eviter l'inhibition de la glucose oxydase lors du dosage du glucose. Deux strategies d'immobilisation du mediateur dans ces capteurs amperometriques sont envisagees, la premiere est fondee sur l'affinite de quelques ferrocenes pour le solvant hydrophobe constitutif du gel, la seconde sur l'affinite de l'oxygene pour les chaines perfluorees du nafion

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting copolymer poly(N-(4-(3-thienyl methylene)-oxycarbonylphenyl)maleimide)-co-pyrrole(P(MBThi-co-Py)). A copolymer was electrochemically synthesized by using sodium dodecyl sulfate (SDS) as supporting electrolyte and characterized by FTIR, scanning electron microscopy (SEM) and conductivity measurements. Immobilization of glucose oxidase (GOD) and polyphenol oxidase (PPO) enzymes were performed in conducting PPy and P(MBThi-co-Py) matrices by electropolymerization. Kinetic parameters, maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes by help of Lineweaver-Burk plot. Effect of temperature and pH on GOD and PPO activity was examined. Operational stability and long term stability of the enzyme electrodes were investigated. The immobilized GOD and PPO electrodes were used for determination of glucose amount in Turkish orange juices and analyzing the concentration of phenolic compounds in Turkish red wines respectively.

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique.Dans ce travail, nous nous sommes intéressés au développement de trois biocapteurs pour la détection de substances biologiquement actives. Le premier permet la détermination simultanée de l’adénosine triphosphate (ATP) et du glucose par ampérométrie, le deuxième celle de la créatine kinase, et le troisième est un biocapteur conductimétique pour la quantification de l’ATP. Dans les deux premiers biocapteurs, deux enzymes (l’hexokinase et la glucose oxydase) sont immobilisées à la surface de microélectrodes constituées d’un disque de platine. Le troisième biocapteur est basé sur l’immobilisation de l’hexokinase sur des microélectrodes interdigitées en or. L’immobilisation est réalisée dans tous les cas par co-réticulation des enzymes en présence d’albumine de sérum bovin à l’aide de glutaraldehyde. Les caractéristiques analytiques des biocapteurs ont été déterminées et différentes procédures ont été développées pour l’analyse d’échantillons réels. Les biocapteurs ont pu être appliqués avec succès à la quantification de l’ATP, du glucose et de la créatine kinase dans des préparations pharmaceutiques et du sérum sanguin
Biosensors are rapid, selective and inexpensive devices that combine a biological recognition element, the so-called bioreceptor (e.g. enzymes, antibodies, DNA or microorganisms) to a physical transducer (e.g. electrochemical, optical, thermal or piezoelectrical). They can be used to detect one specific analyte or one family of analytes for a wide range of applications (e.g. environment, food, health). In this work, the detection of biologically active substances was targeted. A biosensor system for simultaneous determination of adenosine triphosphate (ATP) and glucose, a biosensor for creatine kinase analysis, and a novel conductometric biosensor for ATP determination were developed. In the first two biosensors, two enzymes (hexokinase and glucose oxidase) were immobilized at the surface of platinum disc microelectrodes for amperometric detection. The third biosensor was based on hexokinase immobilized onto gold interdigitated microelectrodes for conductometric detection. In all cases, the enzymes were co-immobilized with bovine serum albumin by cross-linking using glutaraldehyde. Analytical characteristics of the biosensors were determined and different procedures were developed for real samples analysis. The biosensors could be successfully applied to the determination of ATP, glucose, and creatine kinase in pharmaceutical samples and blood serum

Orientador: Jose de Assis Fonseca Faria
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A incorporação e a viabilidade de bactérias probióticas em alimentos ao longo do seu período de estocagem, que resultem em benefício para a saúde do consumidor, é um desafio constante para as indústrias de alimentos, requerendo a compreensão dos fatores intrínsecos e extrínsecos ao processamento. A exposição ao oxigênio apresenta-se como um fator relevante, na medida em que esse grupo microbiano apresenta metabolismo anaeróbio e/ou microaerófio, o que pode resultar em morte celular e perda da funcionalidade do produto. Este trabalho teve o objetivo de avaliar o efeito da glicose oxidase sobre o aumento da sobrevivência das bactérias probióticas em iogurte, bem como em seus parâmetros de qualidade e aceitação sensorial. Foi observado o efeito potencial da glicose oxidase na redução do oxigênio dissolvido no iogurte bem como na manutenção da sua funcionalidade, sem interferência nos parâmetros de qualidade e aceitação sensorial do produto
Abstract: The incorporation and viability of probiotic bacteria in processed food during its storage period is a constant challenge for the food industry, and thus requiring an understanding of intrinsic and extrinsic factors in relation to processing. The oxygen exposure is presented as a relevant factor, since the microbial group presents anaerobic metabolism, which can result in cell death and loss of product functionality. This study aimed to evaluate the effect of glucose oxidase as oxygen absorver for increasing the survival of probiotic bacteria in yogurt as well as its effect on the quality parameters and sensory acceptability. It was noted the potential effect of glucose oxidase in the reduction of dissolved oxygen in yogurt and in maintaining its functionality, without interference on quality parameters and sensory acceptability of the product
Doutorado
Doutor em Tecnologia de Alimentos

Dissertation presented to obtain the Master degree in Biochemistry for Health
"Type 2 diabetes mellitus (T2DM) is one of the biggest health problems in the world. Glucagon like peptide-1, an endogenous gastrointestinal incretin hormone that stimulates insulin secretion, is considered an attractive therapeutic agent for T2DM treatment. Nevertheless, its short half-life has led to the development of longer half-life GLP-1 analogues, as exenatide. Still, current dosage forms for exenatide delivery are not exempt of shortcomings. Nanotechnology-based systems may offer several advantages over conventional dosage forms for drug delivery.(...)"
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Neste trabalho, investigou-se a fabricação e caracterização de filmes ultrafinos de poli(alilamina hidroclorada) PAH com as enzimas glicose oxidase (GOx) e invertase (INV) no desenvolvimento de biossensores amperométricos para a detecção de glicose e sacarose. Filmes nanoestruturados obtidos pela técnica layer-by-layer (LbL) possibilitaram estudar a imobilização de cada enzima juntamente com o polieletrólito, além de diversas arquiteturas bienzimáticas mais complexas, em eletrodos modificados com Azul da Prússia, no desenvolvimento do biossensor. O crescimento dos filmes produzidos foi acompanhado por técnicas de espectroscopia UV-vis e fluorescência; além de técnicas eletroquímicas de voltametria cíclica e amperometria na avaliação das propriedades e resposta do biossensor. Por fim, investigou-se o aprimoramento do biossensor e análises qualitativas preliminares na avaliação de bebidas comerciais
In this work, we investigated the fabrication and characterization of ultrathin films of poly(allylamine hydrochloride) PAH and the enzymes glucose oxidase (GOx) and invertase (INV) in the development of amperometric biosensors for glucose and sucrose detection. Nanostructured films obtained by layer-by-layer (LbL) technique were able to study the immobilization of each enzyme coupled with the polyelectrolyte, beyond different and more complex bienzimatic architectures, in Prussian Blue modified electrodes, for the biosensor development. The films’ growth was monitored by fluorescence and UV-vis spectroscopic techniques; moreover electrochemical techniques such as cyclic voltammetry and amperometry were used on the evaluation of biosensor properties and response. At last, we investigated the biosensor improvement and qualitative initial analysis on the evaluation of commercial beverages

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2016.
The Maillard reaction (MR) involves the condensation reaction between amino acids or proteins with reducing sugars, which occurs commonly in food processing and storage. Maillard reaction products (MRPs) were prepared from glucose-casein model system at pH 8, heated at 60, 75 and 90°C for 6, 12 and 24 h, respectively. Browning intensity (BI) of MRPs, as monitored by absorbance at 420 nm increased with an increase in reaction temperature. The reducing power (RP) of MRPs increased (p < 0.05) as the reaction time increased at 60 and 75°C, while at 90°C an increase in RP was observed from 6 to 12 h and thereafter a slight decrease was observed up to 24 h. The 2,2-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity (ABTS-RS) and Peroxyl radical scavenging (PRS) activity of glucose-casein MRPs produced at 90°C decreased as the reaction time increased. In this study, the ferrous chelation activity of MRPs was higher than that of tert-butylhydroquinone (TBHQ) (0.02%) and Trolox (1 mM), respectively. Moreover, the 1, 1-diphenyl-2-picryl-hydrazil radical scavenging (DPPH-RS) of MRPs increased (p < 0.05) as the reaction time increased irrespective of the heating temperature. The primary and secondary lipid oxidation products were measured using the Peroxide value (PV) and Thiobarbituric acid reactive substance (TBARs) assay in sunflower oil-in-water emulsion, respectively. MRPs derived at 90°C for 12 h had the lowest peroxide value, while the TBARs inhibitory by MRPs ranged from 39.05 – 88.66%. Glucose-casein MRPs displayed superior antioxidant activity than TBHQ (0.02%) and Trolox (1 mM), respectively, as measured by the TBARs assay. The differential scanning calorimetry (DSC) and Rancimat techniques set at 110°C were used to evaluate the oxidative stability the lipid-rich media containing MRPs. At the same temperature program, DSC gave significantly lower reduction times than the Rancimat. Furosine (N-ε-Fructosyl-lysine) and Pyrraline (2-amino-6-(2-formyl-5-hydroxymethyl-1-pyrrolyl)-hexanoic acid) were determined using high pressure liquid chromatography to evaluate the extent of the MR. Furosine concentration of glucose-casein MRPs ranged between 0.44 – 1.075 mg.L-1 in MRPs derived at 60°C, while at 75°C an increase as function of time was observed. MRPs derived at 60 and 75°C exhibited a varied concentration of pyrraline as the reaction time increased with higher temperatures resulted in higher concentrations (0.39 mg.L-1). The results of this study clearly indicated that MRPs possess antioxidant activity and can be used as natural antioxidants in the food industry.

This study investigated the feasibility of oxidative modification with glucose oxidase (GluOx) to enhance the rheological and gelling properties of myofibrillar protein. Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of glucose/FeSO4 compared to a Fenton system (H2O2/FeSO4) were investigated. Firmer and more elastic MP gels were produced by the GluOx-oxidizing system than by the Fenton system at comparable H2O2 levels due to an altered radical reaction pathway. The study further explored the effect of GluOx-mediated oxidation on the efficacy of transglutaminase (TGase) cross-linking of MP in 0.6 M NaCl and the rheological properties of GluOx oxidation/TGase treated MP in MP–lipid emulsion composite gels. The GluOx-mediated oxidation promoted the formation of both soluble and insoluble protein aggregates via disulfide bonds and occlusions of hydrophobic groups. The subsequent TGase treatment converted protein aggregates into highly cross-linked polymers. MP–lipid emulsion composite gels formed with such polymers exhibited markedly enhanced gelling capacity: up to a 4.4-fold increase in gel firmness and a 3.5-fold increase in gel elasticity over untreated protein. Microstructural examination showed small oil droplets dispersed in a densely packed gel matrix when MP was oxidatively modified, and the TGase treatment further contributed to such packing. Comparison of the modification of MP via GluOx oxidation/TGase cross-linking pathway under different salt concentrations (0.3 and 0.6 M NaCl) showed different patterns of MP cross-linking, resulting in different extents of aggregation. Under low-salt condition (0.3 M NaCl), the GluOx/TGase treatment increased the gel strength to the same level as those treated with TGase in 0.6 M NaCl, suggesting a potential application of GluOx/TGase for improving gel strength in low ionic strength conditions. Finally, the application of GluOx oxidation in the development of emulsion-type sausages was studied. The GluOx oxidation/TGase cross-linking improved the textural properties (firmness, chewiness, and rupture force) of emulsion-type sausages under both salt levels (P < 0.05). Under low-salt condition (1.5% NaCl), GluOx/TGase treatment can increase the sausage binding strength to the same level as the control sample under high-salt condition (3% NaCl). The GluOx oxidation/TGase treatment shows promise to improve the textural properties of emulsified meat products. However, the significant decrease of a* value and increase of b* value indicated GluOx-induced color deterioration.

In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS).

Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules.

Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).

Orientador: Marystela Ferreira
Banca: Debora Terezia Balogh
Banca: Débora Gonçalves
O Programa de Pós-Graduação em Ciência e Tecnologia de Materiais, PosMat, tem caráter institucional e integra as atividades de pesquisa em materiais de diversos campi da Unesp
Resumo: Neste trabalho, investigou-se a fabricação e caracterização de filmes ultrafinos de poli(alilamina hidroclorada) PAH com as enzimas glicose oxidase (GOx) e invertase (INV) no desenvolvimento de biossensores amperométricos para a detecção de glicose e sacarose. Filmes nanoestruturados obtidos pela técnica layer-by-layer (LbL) possibilitaram estudar a imobilização de cada enzima juntamente com o polieletrólito, além de diversas arquiteturas bienzimáticas mais complexas, em eletrodos modificados com Azul da Prússia, no desenvolvimento do biossensor. O crescimento dos filmes produzidos foi acompanhado por técnicas de espectroscopia UV-vis e fluorescência; além de técnicas eletroquímicas de voltametria cíclica e amperometria na avaliação das propriedades e resposta do biossensor. Por fim, investigou-se o aprimoramento do biossensor e análises qualitativas preliminares na avaliação de bebidas comerciais
Abstract: In this work, we investigated the fabrication and characterization of ultrathin films of poly(allylamine hydrochloride) PAH and the enzymes glucose oxidase (GOx) and invertase (INV) in the development of amperometric biosensors for glucose and sucrose detection. Nanostructured films obtained by layer-by-layer (LbL) technique were able to study the immobilization of each enzyme coupled with the polyelectrolyte, beyond different and more complex bienzimatic architectures, in Prussian Blue modified electrodes, for the biosensor development. The films' growth was monitored by fluorescence and UV-vis spectroscopic techniques; moreover electrochemical techniques such as cyclic voltammetry and amperometry were used on the evaluation of biosensor properties and response. At last, we investigated the biosensor improvement and qualitative initial analysis on the evaluation of commercial beverages
Mestre

碩士
國立交通大學
生醫工程研究所
103
In recent years, development of biochips and biosensors technology has gained tremendous attentions for its potential in health checks. By integrating the complete micro-analysis system on a chip, biomedical signals can be recorded quickly and then read out by a transducer for further analysis. In order to detect different drugs and biological molecules, different receptors need to be equipped with different sensing end of each molecule; afterwards, the specific analytes reacted with those receptors will be measured via optical or electrical signal by signal converter. However, to avoid the inconvenience of employing various receptors, and to observe a comprehensive environmental changes, we expect pharmaceutical inspection chips capable of detecting a variety of biological molecules or drugs, and the corresponding surrounding temperature and pH values to obtain the optimal operating condittion. Concerning the matters mentioned above, we plan to develop a chip providing information of environmental pH value and temperature under different targets. But in the study, we mainly observe the changing curve of ambient pH value by electrochemical analysis over the traditional printed electrodes. Glucose is employed as the analyte for different pH values, and we obtain the figure of electric current as the function of environmental pH value and glucose concentration, which will play an important role in the development of our biochips. In this study, we conduct the experiment on the printed electrodes rather than ones embedded in glucose oxidase, and the electrochemical analysis is carried out by cyclic voltammetry to find out the preferable voltage. From the results, we find electrons transporting from solution to electrodes be done best by adding electronic substance. However, our chip is not embedded in glucose oxidase, so we add ferrocyanide in the solution to promote the electrons transportation, enhancing the signal detection. Next, quantitative chronoamperometry assay is applied to establish the relationship between analytes conditions and pH value. In this study, we develop an analyzing method in quantifying the influence of glucose concentration and different pH values on the corresponding electric current values. Results have shown that the measured electric current increases with higher glucose concentration, and the neutral state in pH provides a more stable environment for detection condition, of which the measured current changes little. Our results will support the further development of the chips, and the current measurement can be corrected in the practical test results in the future.

Thesis (Ph.D.)--International University Bremen, 2007.
School of Engineering and Science. "Doctor of Philosophy in Biochemical Engineering." Includes bibliographical references (leaves 71-84). Also available online.

碩士
國立屏東技術學院
食品技術研究所
85
The activity of glucose oxidase of Geotrichum sp. No.56 was induced up to 0.451 unit/mg protein by adding 0.75 % sorbitol as a carbon source in synthetic melanoidin. Moreover, the enzyme activity was independent upon the sorbitol concentration. The enzyme specific activvity was up to 0.838 unit/mg protein when the Geotrichum sp. No.56 was cultivated in 500 mL Hinton's flask containing 0.2 % NH4Cl and 0.2 % peptone as nitrogen source and adding 0.1 % KH2PO4 and 0.05% MgSO4*7H2O as salts under the defined condition ( pH 6.0, 125 rpm, at 30 ℃ ) for 6 days. The optimum pH and optimum temperature of the crude enzyme were 6.5 and 50 ℃, respectively.The pH stability of the crude enzyme was 5.5~6.5. The enzyme was thermal stability below 50 ℃. The glucose oxidase was high affinitive to glucose. Furthermore, the inhibitory effect of metal ions (Hg2+, Ag+, Cd2+, Cu2+,Pb2+、、、etc.) and chemical reagents (cysteeeine, IAA, 2-mercaptoethanol,SDS, PCMB, H2O2 and gluconic acid.) on enzyme activity were investigated in the current research. The induced enzyme was suspected as a nonmetalion required enzyme since there was little effect on enzyme activity when EDTA concentration changed. The potential active site of the enzyme was -SHgroup and the end-product inhibition of this enzyme also observed. The crudeglucose oxidase was purified by (NH4)2SO4, DEAE-cellulose and Sephadex G-150column. the optimum pH and optimum temperature of the purified enzyme were6.5 and 45℃, respectively. The pH stability and thermal stability of the purified enzyme were 4.5~6.5 and less than 45 ℃. Th specific activity were upto 7.291 unit/mg and the purified fold was increased to 8.33 folds. The estimated molecular weight of the purified enzyme was 127,500 and 63,500 by Sephadex G-150 and SDS-PAGE, respectively. The enzyme was suggested tobe composed with two subunits having the same molecular weight.

碩士
逢甲大學
纖維與複合材料學系
100
In this study, we successfully combined the advantages of electrospinning technology, gold nanoparticles, graphene and electrochemical biosensor to produce a high-sensitive glucose biosensor. We prepared the nanofiber membranes by electrospun a solution of PVA/GOx/Au or PVA/GOx/Graphene. TEM image indicated that gold nanoparticles and graphene were both well dispersed in nanofiber. We research the effect of content on the sensitivity of biocomposite membranes. The responds time and sensitivity of sample were detected by electrochemical method. And the enzyme activity was detected by UV-Vis Spectroscopy and Beer-Lambert Law. The first additive was gold nanoparticles. The best addition of Gold nanoparticles was 50 ppm in this experiment. The responds time was 5.6 second, and sensitivity was 37.7 μA / mM. In the analysis of enzyme kinetic, the Imax was 434.8 μA, and the Kmapp was 0.83 mM. The enzyme activity was 1.11±0.25 U/mg. Another additive was graphene oxide solution. The sensitivity and enzyme activity of graphene modified PVA/GOx nanofiber membrane increased with the fraction of grapheme increased. The 20 ppm of graphene was the best content in this experiment, with higher sensitivity (38.7μA/mM) and enzyme activity (0.93 ± 0.31 U/mg).

碩士
元智大學
光電工程研究所
99
In this study, we demonstrated a full-field heterodyne interferometer to analyze two dimensional (2D) dynamic phase variation of the chemical reaction between the testing sample and the glucose sensor. Because of high selectivity of the glucose oxidase (GOx), we can ensure the phase variation coming from the reaction between GOx and glucose dissolved in the complicated testing sample. Based on the advantages of the full-field heterodyne interferometer, we can investigate the 2D reaction behavior of the GOx under different immobilized pH value and concentration. According to our results, the best response time is shorter than 2.2 seconds observed in this study. We also obtained the calibration curve with high linearity within the glucose concentration of 100 ~ 600 mg/dL. Finally, we can get the resolution of this method can be reached to 39.286 mg/dL.

碩士
國立中興大學
生醫工程研究所
103
Compared to traditional multi-wall carbon nanotube (MWCNT)/glucose oxidase (GOx) composite sensors, a novel carbon heterostructure nanomaterial of multi-wall carbon nanotube@graphene oxide nanoribbon (MWCNT@GONR)presented the better sensing properties for glucose detection. The optimal ratio of MWCNT@GONR to GOx was 1:1 with the largest electron transfer rate. Furthermore, we developed a dielectrophoretic (DEP) technique to fix the MWCNT/GOx composite on the thin-film gold electrodes. The method has promising potential to absorb the MWCNT/GOx composite on the surface of electrodes for constructing a more effective third-generation glucose biosensor.