La present tesi doctoral recull l'estudi de diverses modificacions químiques de sensors amperomètrics, adreçades cap a cercar solucions per als problemes típicament implícits en el desenvolupament de biosensors basats en enzims oxidoreductases, alhora que es busca la compatibilitat d'aquestes modificacions amb les estratègies d'immobilització enzimàtica emprades.La primera part del treball inclou el desenvolupament de quimiosensors reproduïbles i estables per a la determinació del cofactor NADH, del qual depenen els enzims deshidrogenases, i del compost peròxid d'hidrogen, obtingut com a producte de les reaccions en què participen gran part dels enzims oxidases. Els quimiosensors per a la determinació de NADH s'han basat en l'ús de diferents mediadors d'oxidació-reducció, mitjançant vàries estratègies d'incorporació als sistemes de detecció amperomètrica (en solució, per adsorció sobre la superfície electròdica o en membranes de Nafió, incorporats en matrius polimèriques de compòsits de grafit-epoxi, electropolimeritzats o immobilitzats en membranes de polisulfona). Els quimiosensors basats en membranes de polisulfona han mostrat nombrosos avantatges respecte la resta de quimiosensors desenvolupats. De fet, la polisulfona es presenta en aquest estudi com a un bon material polimèric per al desenvolupament de quimiosensors amperomètrics, atès que aconsegueix evitar els problemes de passivació de la superfície electròdica implícits en la determinació del cofactor NADH, i al mateix temps permet una retenció excel·lent dels mediadors immobilitzats al seu interior, amb una absència total de pèrdues d'aquestes espècies per dissolució. D'altra banda, en relació al desenvolupament de quimiosensors per a la determinació de peròxid d'hidrogen, s'han sintetitzat gels de sílice que incorporen diferents metalls que actuen com a catalitzadors dels processos d'oxidació i reducció del peròxid d'hidrogen. Aquests gels s'han dipositat com a mescles del corresponent xerogel amb acetat de cel·lulosa i polietilenglicol, a fi d'aconseguir membranes que minimitzen les limitacions d'aquest tipus de materials (formació d'esquerdes, absorció d'aigua,...). S'han emprat diferents tècniques d'anàlisi amb l'objectiu de dur a terme estudis de caracterització dels quimiosensors basats en membranes de polisulfona i en xerogels modificats amb metalls.La segona part del treball es va dedicar al desenvolupament de biosensors basats en diversos enzims oxidoreductases, mitjançant l'ús dels quimiosensors prèviament desenvolupats. Els quimiosensors per a NADH s'han adaptat per al desenvolupament de biosensors per a lactat, basats en la incorporació de l'enzim L-lactat deshidrogenasa en matrius de gels de sílice i en membranes de polisulfona, i de biosensors per a ió amoni, basats en la incorporació de l'enzim glutamat deshidrogenasa en polímers de mediador o en membranes de polisulfona. També es va desenvolupar un biosensor bienzimàtic per a la determinació d'urea, basat en la incorporació dels enzims glutamat deshidrogenasa i ureasa en membranes de polisulfona. D'altra banda, els quimiosensors per a peròxid d'hidrogen s'han emprat per al desenvolupament de biosensors per a glucosa, basats en la incorporació de l'enzim glucosa oxidasa en gels de sílice mitjançant vàries configuracions diferents. Els biosensors desenvolupats han demostrat la capacitat de les membranes de polisulfona i dels gels de sílice per a incorporar enzims. A més, en alguns casos, com el biosensor per a ió amoni, s'han aconseguit unes característiques analítiques excel·lents (sensibilitat elevada, intervals lineals amplis, temps de resposta curts, bona reproductibilitat entre corbes de calibració successives,...).Finalment, la darrera part del treball es va basar en l'adaptació dels quimiosensors i biosensors desenvolupats, basats en una configuració cilíndrica, a una configuració plana, mitjançant processos de serigrafia, i la seva posterior implementació en sistemes de flux. / The aim of this work was the study of different chemical modifications of amperometric sensors in order to minimise the problems involved in the development of biosensors based on oxidoreductase enzymes, trying to find compatibility between the used chemical modifications and the employed enzymatic immobilisation strategies.The first part of the work was devoted to the development of reliable and stable chemosensors for the determination of NADH cofactor and hydrogen peroxide, for the further development of dehydrogenase- and oxidase-based biosensors, respectively, since dehydrogenase enzymes are NAD-dependent and most of the oxidase enzymes involve hydrogen peroxide as a reaction product. Chemosensors for the determination of NADH were based on the incorporation of different electron mediators, using several incorporation strategies into the amperometrical detection system (in solution, by adsorption onto the electrode surface or onto Nafion membranes, by incorporation inside polymeric matrices of graphite-epoxy composites, by electropolymerization or by immobilisation inside polysulfone membranes). Chemosensors based on polysulfone membranes have shown many advantages in front of the other developed chemosensors. In fact, polysulfone is presented for the first time as an adequate polymeric material for the development of amperometric chemosensors, since it avoids the fouling surface problems typically involved in the amperometric determination of NADH cofactor, while at the same time allows an excellent retention of the immobilised mediators inside the membrane, without leakage of the immobilised species into the solution. On the other hand, in relation to the development of chemosensors for the determination of hydrogen peroxide, several sol-gels have been synthesised, which incorporate different metals acting as catalysts for the oxidation and reduction processes of hydrogen peroxide. These sol-gels have been mixed with cellulose acetate and polyethyleneglycol in order to be deposited as membranes, minimising the limitations of this kind of materials (cracking, water absorption,...). Different analysis techniques have been used with the aim of characterising the final chemosensors based on polysulfone membranes and metal-modified-xerogels. The second part of the work was directed towards the development of biosensors based on different oxidoreductase enzymes, using the previously developed chemosensors. Chemosensors for NADH have been used for developing lactate biosensors, based on the incorporation of L-lactate dehydrogenase enzyme inside sol-gel matrices and polysulfone membranes, and ammonium biosensors, based on the entrapment of glutamate dehydrogenase enzyme inside electropolymerized mediators or polysulfone membranes. Furthermore, a urea bienzymatic biosensor was developed, based on the incorporation of glutamate dehydrogenase and urease enzymes inside polysulfone membranes. On the other hand, chemosensors for hydrogen peroxide have been used for developing glucose biosensors, based on the immobilisation of glucose oxidase enzyme in sol-gels using different configurations. Both strategies, based on polysulfone membranes and sol-gels, have shown the ability of these membranes to incorporate enzymes into the biosensor configuration. Additionally, some of the developed biosensors, such as the ammonium biosensor, have achieved excellent analytical characteristics (high sensitivity, wide linear ranges, fast response times, good reproducibility among successive calibration curves,...).Finally, the last part of this work was based on the application of screen-printing technology for the preparation of the developed chemosensors and biosensors with a planar configuration, and their further implementation in flow systems.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 243-271). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 155-168).

Corticosterone in foetal tissues after injection of the mother with ¹⁴C-corticosterone was determined by acetylation. with ³H-acetic anhydride and crystallization to constant specific activity. The corticosterone content of whole foetal tissue varied between gestational days 13 and 17 from 641 to 300 ng/g respectively. The specific activity of foetal hormone recovered remained essentially constant; after a 15-minute pulse this was as much as one-fourth that of maternal hormone. However, placenta, head and liver showed distinctly different patterns of metabolism, which changed greatly during this time in head and liver, with a decrease in the conversion of corticosterone to 11-dehydrocorticosterone and a rise in foetal liver 113-hydroxysteroid:NADP oxidoreductase activity. This mitochondrial enzyme, Km=33yM, pH optimum 6, which reduces the 11-dehydro metabolite to the biologically active 116-OH compound, increased sharply, raising the relative amount of the latter in foetal tissues from 15 to 91% during this period. One day after removal of maternal adrenals, foetal corticosterone was normal and maternal levels close to normal, indicating ability of foetal adrenals to function. Maternal hormone, however, crossed to the foetus readily and it was considered most likely that, normally, the maternal source predominates. Regardless of origin, foetal or maternal, however, the hormone is maintained in different foetal tissues in a distinct and different manner. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Pyridine nucleotide transhydrogenase catalyzes the reversible transfer of hydride ion equivalents between NADP(H) and NAD(H). In this study, the activity of the enzyme was measured by following the rate of reduction of an analogue of NAD⁺ , 3-acetylpyridxne-NAD⁺ (APNAD⁺ ) by NADPH. The enzyme was solubilized by detergents such as lysolecithin, sodium cholate (in the presence of ammonium sulphate) or Triton X-100. The molecular size of the solubilized enzyme was examined using sucrose density gradient centrifugation in the presence of Brij 58. These detergents gave soluble fragments of different sizes. That solubilized by Triton X-100 or sodium cholate (in the presence of ammonium sulphate) existed as large aggregates with sedimentation coefficients of 24.5 to 25.4S, whereas that obtained with lysolecithin consisted mainly of a species with a sedimentation coefficient of 7.3 to 16.5S. The fragment resulting from the solubilization with Triton X-100 could be cleaved into a smaller species (8.4S) by lysolecithin. Analysis by chromatography on Sepharose 6B of the enzyme preparation solubilized by sodium cholate (in the presence of ammonium sulphate), revealed the presence of other constituents of the membrane, such as succinate dehydrogenase, ATPase and cytochrome b₁. The molecular weight of the aggregate was estimated to be between 0.25 x 10⁶ and 4 x 10⁶. The enzyme in this preparation could not be further disaggregated by Tween 80, Brij 3 5 or Triton X-100. Chromatography of this preparation on DEAE-Sepharose CL-6B yielded a maximum purification of 37 to 68-fold over that of the membrane particle suspension. The specific activity of the enzyme was 8.8 to 15.7 umol per min per mg protein. Analysis of the partially purified enzyme on poly-acrylamide gels in the presence of sodium dodecyl sulphate revealed enrichment of several major polypeptide bands of molecular weights 90 000, 57 000, 50 000 and 40 000, coinciding with the transhydrogenase activity. The partially purified enzyme could be activated by detergents of the Tween or Brij series and by lysolecithin, palmitic acid and phospholipid extracts from E. coli. Measurements of the steady-state kinetics of the membrane-bound enzyme gave values of 45.6 and 106.7 uM for the substrates APNAD+ and NADPH, and dissociation constants of 3.6 and 16.2 uM, respectively. Lineweaver-Burk plots for each substrate at different fixed concentrations of the other substrate revealed a unique pattern of lines that is characteristic of rapid equilibrium random bireactant mechanisms with two dead-end products. In this type of mechanism each substrate is able to interact at the binding site of the other substrate to cause inhibition of enzyme activity. This mechanism was confirmed by kinetic studies using the alternate substrates deamino-NADPH and NAD⁺ , as well as by product inhibitxon studies. The adenine nucleotides 5’-AMP and ADP were competitive inhibitors of the APNAD+-binding site, while 2'-AMP was a competitive inhibitor of the NADPH-binding site on the enzyme. Studies on the active site using 2,3-butanedione or phenyl glyoxal revealed the presence of one modifiable arginyl residue per active site on the enzyme. Protection against modification by 2,3-butanedione was afforded by 2'-AMP, 5'-AMP, NAD+ and NADP+. Inhibition by 2,3-butanedione was enhanced in the presence of low concentrations of NADH or NADPH suggesting that binding of the reduced pyridine nucleotides, possibly at an allosteric site, causes a conformational change in the enzyme. Enhancement of in-activation of the enzyme by TPCK-trypsin was also observed in the presence of reduced pyridine nucleotides. NAD(P)H was oxidized by 2,3-butanedione in the presence of light. The rate of photooxidation was greatest at pH 7 and when the wavelength of incident light was 410 nm. This indicates that absorption of light by the diketone was necessary for the occurrence of the photooxidation reaction. The stochiometry of the reaction between NADH and 2,3-butanedione was 1:1. The possible nature of the reaction product is discussed in the thesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Aspects of recombinant enzyme production were characterized in genetically engineered Escherichia coli. Five strains transformed with the xylose isomerase overproduction system (pRK248/pTXI-1) were compared, based on parameters of cell metabolism and inducible enzyme activity in shake flask cultures. E. coli strain LE392 (pRK248/pTXI-l) performed the best with respect to nearly all of the parameters tested. The stability of the overproducing plasmids was tested in prolonged serial shake flask cultures. Segregational instability occurred in cultures lacking antibiotic selective pressure. The onset of this instability was influenced by host strain, plasmid construct and cultivation media. Despite its occurrence, the degree of plasmid instability, even in the worst case, would not be detrimental in the proposed application of this overproduction system. The cell mass and enzyme production of strain LE392 (pRK248/pTXI-1) was characterized in four batch cultures. It was determined that both carbon source starvation and the accumulation of acetate as a byproduct of glucose metabolism diminished the E. coli's ability to produce xylose isomerase. The peak enzyme production was determined to be 1320 International Units (IU) of xylose isomerase activity per liter of culture. The cell mass and enzyme production of strain LE392 (pRK248/pTXI-l) was studied in fed-batch cultures. The amount of xylose isomerase the cells produced was dependent on the degree of glucose limitation they experienced during cultivation. The peak enzyme production was 3250 IU/L. The results of these experiments were interpreted in the context of this overproduction system and its proposed application for the production of ethanol from plant biomass. / Ph. D.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.