Estabilidad de la glucosa oxidasa en sistemas amorfos formados por los disacáridos sacarosa, maltosa y trehalosa

Glucose-oxidase (GOD), suffers conformational change during freeze-drying. In order to determine the protection level granted by amorphous matrices (AM) of saccharose, maltose, trehalose and their combinations, the thermal inactivation constants (K D) of GOD trapped in these systems were determined. For its evaluation, GOD samples were balanced at different water activities and heated up to 30, 50 and 70 ºC. The best AM found for GOD stability was saccharose-trehalose (5/10% p/v). The K D values (K D.10-4) at a w = 0.0 were 3 at 30 ºC and 6 at 70 ºC. For non-protected GOD under the same conditions these values were 48 at 30 ºC and 257 at 70 ºC.

Printed platforms for paper-based analytical applications

Paper-based analytical technologies enable quantitative and rapid analysis of analytes from various application areas including healthcare, environmental monitoring and food safety. Because paper is a planar, flexible and light weight substrate, the devices can be transported and disposed easily. Diagnostic devices are especially valuable in resourcelimited environments where diagnosis as well as monitoring of therapy can be made even without electricity by using e.g. colorimetric assays. On the other hand, platforms including printed electrodes can be coupled with hand-held readers. They enable electrochemical detection with improved reliability, sensitivity and selectivity compared with colorimetric assays. In this thesis, different roll-to-roll compatible printing technologies were utilized for the fabrication of low-cost paper-based sensor platforms. The platforms intended for colorimetric assays and microfluidics were fabricated by patterning the paper substrates with hydrophobic vinyl substituted polydimethylsiloxane (PDMS) -based ink. Depending on the barrier properties of the substrate, the ink either penetrates into the paper structure creating e.g. microfluidic channel structures or remains on the surface creating a 2D analog of a microplate. The printed PDMS can be cured by a roll-ro-roll compatible infrared (IR) sintering method. The performance of these platforms was studied by printing glucose oxidase-based ink on the PDMS-free reaction areas. The subsequent application of the glucose analyte changed the colour of the white reaction area to purple with the colour density and intensity depending on the concentration of the glucose solution. Printed electrochemical cell platforms were fabricated on paper substrates with appropriate barrier properties by inkjet-printing metal nanoparticle based inks and by IR sintering them into conducting electrodes. Printed PDMS arrays were used for directing the liquid analyte onto the predetermined spots on the electrodes. Various electrochemical measurements were carried out both with the bare electrodes and electrodes functionalized with e.g. self assembled monolayers. Electrochemical glucose sensor was selected as a proof-of-concept device to demonstrate the potential of the printed electronic platforms.

ENZYMATIC DETERMINATION OF STARCH IN DOCE DE LEITE USING DIALYSIS

The importance of starch for the food industry makes it necessary to develop new, fast, economic and accurate methodologies for its quantification. In the present paper starch hydrolysis using commercial enzymes of industrial grade are studied aiming to develop an easy and cheap analysis, available to a greater number of industries and technicians. The proposed method is simple, divided in a first step where soluble sugars are eliminated from the samples by using dialysis, followed by starch hydrolysis of the retained fraction with a thermoresistent bacterial alfa-amylase (Termamyl 120L®) and an amyloglucosidase (AMG 300L®). The hydrolysis conditions were those suggested by the enzyme producer. After the hydrolysis step the material was dialysed again for the extraction of glucose that was quantified by the glucose-oxidase colorimetric reactant. The results allowed the construction of calibration equations for starch determination on the analyzed samples. These samples were produced on a laboratory scale and native and acid-modified corn starches were added in known concentrations. By considering the final dilutions employed for glucose determination on the samples, it was possible to confirm that they were identical to that of the glucose-oxidase reactant calibration.

Development of an enzyme immobilization platform based on microencapsulation for paper-based biosensors

Un papier bioactif est obtenu par la modification d’un papier en y immobilisant une ou plusieurs biomolécules. La recherche et le développement de papiers bioactifs est en plein essor car le papier est un substrat peu dispendieux qui est déjà d’usage très répandu à travers le monde. Bien que les papiers bioactifs n’aient pas connus de succès commercial depuis la mise en marche de bandelettes mesurant le taux de glucose dans les années cinquante, de nombreux groupes de recherche travaillent à immobiliser des biomolécules sur le papier pour obtenir un papier bioactif qui est abordable et possède une bonne durée de vie. Contrairement à la glucose oxidase, l’enzyme utilisée sur ces bandelettes, la majorité des biomolécules sont très fragiles et perdent leur activité très rapidement lorsqu’immobilisées sur des papiers. Le développement de nouveaux papiers bioactifs pouvant détecter des substances d’intérêt ou même désactiver des pathogènes dépend donc de découverte de nouvelles techniques d’immobilisation des biomolécules permettant de maintenir leur activité tout en étant applicable dans la chaîne de production actuelle des papiers fins. Le but de cette thèse est de développer une technique d’immobilisation efficace et versatile, permettant de protéger l’activité de biomolécules incorporées sur des papiers. La microencapsulation a été choisie comme technique d’immobilisation car elle permet d’enfermer de grandes quantités de biomolécules à l’intérieur d’une sphère poreuse permettant leur protection. Pour cette étude, le polymère poly(éthylènediimine) a été choisi afin de générer la paroi des microcapsules. Les enzymes laccase et glucose oxidase, dont les propriétés sont bien établies, seront utilisées comme biomolécules test. Dans un premier temps, deux procédures d’encapsulation ont été développées puis étudiées. La méthode par émulsion produit des microcapsules de plus petits diamètres que la méthode par encapsulation utilisant un encapsulateur, bien que cette dernière offre une meilleure efficacité d’encapsulation. Par la suite, l’effet de la procédure d’encapsulation sur l’activité enzymatique et la stabilité thermique des enzymes a été étudié à cause de l’importance du maintien de l’activité sur le développement d’une plateforme d’immobilisation. L’effet de la nature du polymère utilisé pour la fabrication des capsules sur la conformation de l’enzyme a été étudié pour la première fois. Finalement, l’applicabilité des microcapsules de poly(éthylèneimine) dans la confection de papiers bioactifs a été démontré par le biais de trois prototypes. Un papier réagissant au glucose a été obtenu en immobilisant des microcapsules contenant l’enzyme glucose oxidase. Un papier sensible à l’enzyme neuraminidase pour la détection de la vaginose bactérienne avec une plus grande stabilité durant l’entreposage a été fait en encapsulant les réactifs colorimétriques dans des capsules de poly(éthylèneimine). L’utilisation de microcapsules pour l’immobilisation d’anticorps a également été étudiée. Les avancées au niveau de la plateforme d’immobilisation de biomolécules par microencapsulation qui ont été réalisées lors de cette thèse permettront de mieux comprendre l’effet des réactifs impliqués dans la procédure de microencapsulation sur la stabilité, l’activité et la conformation des biomolécules. Les résultats obtenus démontrent que la plateforme d’immobilisation développée peut être appliquée pour la confection de nouveaux papiers bioactifs.

The role of carbohydrates in seed germination and seedling establishment of Himatanthus sucuuba, an Amazonian tree with populations adapted to flooded and non-flooded conditions

Background and Aims In the Amazonian floodplains plants withstand annual periods of flooding which can last 7 months. Under these conditions seedlings remain submerged in the dark for long periods since light penetration in the water is limited. Himatanthus sucuuba is a tree species found in the `varzea` (VZ) floodplains and adjacent non-flooded `terra-firme` (TF) forests. Biochemical traits which enhance flood tolerance and colonization success of H. sucuuba in periodically flooded environments were investigated. Methods Storage carbohydrates of seeds of VZ and TF populations were extracted and analysed by HPAEC/PAD. Starch was analysed by enzyme (glucoamylase) degradation followed by quantification of glucose oxidase. Carbohydrate composition of roots of VZ and TF seedlings was studied after experimental exposure to a 15-d period of submersion in light versus darkness. Key Results The endosperm contains a large proportion of the seed reserves, raffinose being the main nonstructural carbohydrate. Around 93% of the cell wall storage polysaccharides (percentage dry weight basis) in the endosperm of VZ seeds was composed of mannose, while soluble sugars accounted for 2.5%. In contrast, 74% of the endosperm in TF seeds was composed of galactomannans, while 22% of the endosperm was soluble sugars. This suggested a larger carbohydrate allocation to germination in TF populations whereas VZ populations allocate comparatively more to carbohydrates mobilized during seedling development. The concentration of root non-structural carbohydrates in non-flooded seedlings strongly decreased after a 15-d period of darkness, whereas flooded seedlings were less affected. These effects were more pronounced in TF seedlings, which showed significantly lower root non-structural carbohydrate concentrations. Conclusions There seem to be metabolic adjustments in VZ but not TF seedlings that lead to adaptation to the combined stresses of darkness and flooding. This seems to be important for the survival of the species in these contrasting environments, leading these populations to different directions during evolution.

Enzyme immobilisation on electroactive nanostructured membranes (ENM): Optimised architectures for biosensing

Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyanoferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H(2)O(2), the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 mu M and an apparent Michaelis-Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples. (C) 2008 Elsevier B.V. All rights reserved.

Using phospholipid Langmuir and Langmuir-Blodgett films as matrix for urease immobilization

The immobilization of enzymes in organized two-dimensional matrices is a key requirement for many biotechnological applications. In this paper, we used the Langmuir-Blodgett (LB) technique to obtain controlled architectures of urease immobilized in solid supports, whose physicochemical properties were investigated in detail. Urease molecules were adsorbed at the air-water interface and incorporated into Langmuir monolayers of the phospholipid dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation of urease made DPPG monolayers more flexible and caused the reduction of the equilibrium and dynamic elasticity of the film. Urease and DPPG-urease mixed monolayers could be transferred onto solid substrates, forming LB films. A close packing arrangement of urease was obtained, especially in the mixed LB films, which was inferred with nanogravimetry and electrochemistry measurements. From the blocking effect of the LB films deposited onto indium tin oxide (ITO) substrates, the electrochemical properties of the LB films pointed to a charge transport controlled by the lipid architecture. (c) 2007 Elsevier Inc. All rights reserved.

Development of Highly Selective Enzymatic Devices Based on Deposition of Permselective Membranes on Aligned Nanowires

We describe a simple and efficient strategy to fabricate enzymatic devices based on the deposition of glucose oxidase on aligned and highly oriented CoNiMo metallic nanowires. CoNiMo nanowires with an average diameter of 200 nm and length of 50 mu m were electrodeposited on Au-covered alumina substrates via electrodeposition, using alumina membranes as templates. Enzyme-modified electrodes were fabricated via enzyme immobilization using a cross-linker. To minimize nonspecific reactions in the presence of interfering agents, a permselective membrane composed of poly(vinylsulfonic acid) and polyamidoamine dendrimer was deposited via electrostatic interaction. The formation of hydrogen peroxide as a product of the enzymatic reaction was monitored at low overpotential, 0.0 V (vs Ag/AgCl). The detection limit was estimated at 22 mu M under an applied potential of 0.0 V. The apparent Michaelis-Menten constant determined from the Lineweaver-Burke plot was 2 mM.

Copper hexacyanoferrate nanoparticles modified electrodes: A versatile tool for biosensors

Copper hexacyanoferrate nanoparticles of about 30 nm in size have been prepared by the sonochemical irradiation of a mixture of aqueous potassium ferricyanide and copper chloride solutions. The nanoparticles were immobilized onto fluorine doped tin oxide (FTO) electrodes by using the electrostatic deposition layer-by-layer technique (LbL), obtaining electroactive films with electrocatalytic properties towards H2O2 reduction, providing higher currents than those observed for electrodeposited bulk material, even in electrolytes containing NH4+, Na+ and K+. The nanoparticles assembly was used as mediator in a glucose biosensor by immobilizing glucose oxidase enzyme by both, cross-linking and LbL. techniques. Sensitivities obtained were dependent on the immobilization method ranging from 1.23 mu A mmol(-1) L cm(-2) for crosslinking to 0.47 mu A mmol(-1) L cm(-2) for LbL; these values being of the same order than those obtained with electrodes where the amount of enzyme used is much higher. Moreover, the linear concentration range where the biosensors can operate was 10 times higher for electrodes prepared with the LbL immobilization method than with the conventional crosslinking one. (C) 2008 Elsevier B.V. All rights reserved.

Immobilization of Catalysts of Biological Interest on Porous Oxidized Silicon Surfaces

The present paper deals with the immobilization of redox mediators and proteins onto protected porous silicon surfaces to obtain their direct electrochemical reactions and to retain their bioactivities. This paper shows that MP-11 and viologens are able to establish chemical bonds with 3-aminopropyltriethoxylsilane-modified porous silicon surface. The functionalization of the surfaces have been fully characterized by energy dispersive X-ray analysis (EDX) and X-ray photoelectron spectroscopy (XPS) to examine the immobilization of these mediators onto the solid surface. Amperometric and open circuit potential measurements have shown the direct electron transfer between glucose oxidase and the electrode in the presence of the viologen mediator covalently linked to the 3-aminopropyltriethoxylsilane (APTES)-modified porous silicon surfaces.

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.

Contribuição da glicemia pós-desjejum para o controle glicêmico do paciente com diabetes melito tipo 2

Estudos epidemiológicos observaram que glicemias pós-prandiais (GPPs) elevadas são fator principal na ocorrência de doenças cardiovasculares. Sabe-se que a hemoglobina glicada (HbA1C) reflete a glicemia média dos últimos 2-3 meses, entretanto é controversa a contribuição relativa da glicemia de jejum (GJ) e GPP para o valor da HbA1C. OBJETIVO: Avaliar a contribuição da GJ e GPPs para o valor da HbA1C em pacientes com diabetes melito tipo 2 (DM2). MÉTODOS: Participaram 53 indivíduos com DM2, estáveis e em tratamento com antidiabéticos orais (n= 27) e/ou insulina (n= 26). Cada paciente comparecia a 3 visitas a intervalos de 2 meses. em cada visita era medida a GJ, as GPPs (2h pós-desjejum: GPD e pós-almoço: GPA) e a HbA1C, sendo fornecido o desjejum e o almoço segundo seus hábitos alimentares. Mediu-se a glicose plasmática pela glicose-oxidase e a HbA1C, pela cromatografia de troca iônica. Realizou-se a análise das associações pelo coeficiente de correlação de Spearman, com P< 0,05. RESULTADOS: A HbA1C correlacionou-se melhor em cada visita ao longo do estudo com a GPD (r: 0,66­0,48), a glicemia média (r: 0,64­0,41), a área abaixo da curva glicêmica (r : 0,64­0,46) e a GPP média (r: 0,59­0,41). CONCLUSÕES: A GPD mostrou-se um parâmetro eficaz adicional no monitoramento glicêmico dos pacientes com DM2.

Periodontal Disease Decreases Insulin Sensitivity and Insulin Signaling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.)

The protein complement of the secretion from hypopharyngeal gland of nurse-bees (Apis mellifera L.) was partially identified by using a combination of 2D-PAGE, peptide sequencing by MALDI-PSD/MS and a protein engine identification tool applied to the honeybee genome. The proteins identified were compared to those proteins already identified in the proteome complement of the royal jelly of the honey bees. The 2D gel electrophoresis demonstrated this protein complement is constituted of 61 different polypepides, from which 34 were identified as follows: 27 proteins belonged to MRJPs family, 5 proteins were related to the metabolism of carbohydrates and to the oxido-reduction metabolism of energetic Substrates, I protein was related to the accumulation of iron in honeybee bodies and I protein may be a regulator of MRJP-1 oligomerization. The proteins directly involved with the carbohydrates and energetic metabolisms were: alpha glucosidase, glucose oxidase and alpha amylase, whose are members of the same family of enzymes, catalyzing the hydrolysis of the glucosidic linkages of starch; alcohol dehydrogenase and aldehyde dehydrogenase, whose are constituents of the energetic metabolism. The results of the present manuscript support the hypothesis that the most of these proteins are produced in the hypoharyngeal gland of nurse-bees and secreted into the RJ. (C) 2004 Elsevier Ltd. All rights reserved.

Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)