"Development and applications of enzymatic and microbial biosensors"

Se construyeron biosensores enzimáticos cableados basados en la inmovilización de una oxidasa (glucosa oxidasa, lactato oxidasa ó peroxidasa) en un hidrogel redox formado por el entrecruzamiento de la enzima, polialilamina conteniendo un cornplejo de osmio (PAAOs) y polietilediglicidiléter (PEG) como entrecruzante, sobre electrodos de carbón vítreo y de oro. Se estudió el rango de linealidad y la reproducibilidad de estos electrodos al ser utilizados como detectores en un sistema FIA. Utilizando electrodos basados en glucosa oxidasa se estudió el efecto de distintas proporciones de entrecruzante (PEG); utilizando técnicas de voltametrías cíclicas se estudió el efecto del cambio de fuerza iónica en el medio. La leche afecta de manera irreversible a estos electrodos monoezimáticos. Se diseñaron distintos tipos de biosensores bienzimáticos bicapa, en los que peroxidasa entrecruzada con PAAOs se encuentra en la capa mas cercana al electrodo, y lactato oxidasa en la capa más externa. Se estudió el efecto de la leche sobre ambas capas enzimáticas, demostrandose que esta configuración es más apropiada para la determinación de la concentración de L-lactato en muestras Iácteas. Se ha descripto un método rápido para determinar la capacidad fermentativa de distintas especies de Lactobacillus. Este método permite cuantificar esta capacidad, proporcionando una información mas completa del metabolismo de los hidratos de carbono; posibilita el estudio de cepas mutantes suministrando inforrnación más completa acerca de las posibles modificaciones en el metabolismo de los fuentes de carbono utilizadas.Enzymatic "wired" biosensors based in the immobilization of one oxidase (glucose oxidase, lactate oxidase or peroxidase) within an redox hydrogel formed by crosslinking of enzyme, polyallylamine containing an osmium complex (PMOS) and poly(ethy1ene glycol) (PEG), onto glassy carbon or gold electrodes were constructed. We study the reproducibility and lineal span of these electrodes when they were used as detectors in a FIA system. To complete their characterization we study the effect of different PMOS 1 PEG ratios in the construction of enzymatic biosensores, the effect of pH, temperature and ionic strength. The effect of milk samples and their response were analyzed, these complex analytical matrix affect these electrodes catalytic current. We designs bilayer bienzyme biosensors; in these system the peroxidase layer, over the electrode ("wired"), are separated from the outer membrane containing lactate oxidase (not "wired1'). We study the effect over both layer of milk samples; from these studies a bienzyme electrode configuration was found as the most appropriate array from the determination of L-lactate in milk samples. A microbial biosensor based on calcium alginate immobilized Lactobacillus cells coupled to a pH electrode was developed for quantitative determination of carbohydrate fermentation activity. The evolution of acid production in a continuous flow-stopped biorreactor was monitored for different sugar solution. The procedure can give quantitative results compared to other reported techniques for carbohydrate fermentation pattern from which only qualitative results are obtained.Fil:Cortón, Eduardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Development of a Novel Method for in vivo Determination of Activation Energy of Glucose Transport Across S. cerevisiae Cellular Membranes. A Biosensor-like Approach

Whereas biosensors have been usually proposed as analytical tools, used to investigate the surrounding media pursuing an analytical answer, we have used a biosensor-like device to characterize the microbial cells immobilized on it. We have studied the kinetics of transport and degradation of glucose at different concentrations and temperatures. When glucose concentrations of 15 and 1.5 mM were assayed, calculated activation energies were 25.2 and 18.4 kcal mol−1, respectively, in good agreement with previously published data. The opportunity and convenience of using Arrhenius plots to estimate the activation energy in metabolic-related processes is also discussed

Microbial Fuel Cells Applied to the Metabolically-Based Detection of Extraterrestrial Life

Since the 1970's, when the Viking spacecrafts carried out experiments aimed to the detection of microbial metabolism on the surface of Mars, the search for nonspecific methods to detect life in situ has been one of the goals of astrobiology. It is usually required that the methodology can detect life independently from its composition or form, and that the chosen biological signature points to a feature common to all living systems, as the presence of metabolism. In this paper we evaluate the use of Microbial Fuel Cells (MFCs) for the detection of microbial life in situ. MFCs are electrochemical devices originally developed as power electrical sources, and can be described as fuel cells in which the anode is submerged in a medium that contains microorganisms. These microorganisms, as part of their metabolic process, oxidize organic material releasing electrons that contribute to the electric current, which is therefore proportional to metabolic and other redox processes. We show that power and current density values measured in MFCs using microorganism cultures or soil samples in the anode are much larger than those obtained using a medium free of microorganisms or sterilized soil samples, respectively. In particular, we found that this is true for extremophiles, usually proposed to live in extraterrestrial environments. Therefore, our results show that MFCs have the potential to be used to detect microbial life in situ.Comment: To be published in Astrobiolog

Immune biomarkers in older adults: role of physical activity

3rd International Conference on Occupational & Environmental Toxicology/3rd Ibero-American Meeting on Toxicology and Environmental Health International, 21-23 June 2016, Porto, Portugal[Abstract] Aging is associated with a decline in the normal functioning of the immune system. Several studies described the relationship between immunological alterations, including immunosenescence and inflammation, and aging or age-related outcomes, such as sarcopenia, depression, and neurodegenerative disorders. Physical activity is known to improve muscle function and to exert a number of benefits on older adult health, including reduced risk for heart and metabolic system chronic diseases. However, the positive influence of physical activity on the immune system has not been elucidated. In order to shed light on the role of physical activity in immune responses of older individuals, a number of immunological parameters comprising % lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, and CD16+56+) and serum levels of neopterin and tryptophan metabolism products were evaluated in peripheral blood samples of older adults performing normal (N = 170) or reduced (N = 89) physical activity. In addition, the potential influence of other clinical and epidemiological factors was also considered. Results showed that subjects with reduced physical activity displayed significantly higher levels of CD4+/CD8+ ratio, kynurenine/tryptophan ratio, and serum neopterin, along with lower %CD19+ cells and tryptophan concentrations. Further, some immunological biomarkers were associated with cognitive impairment and functional status. These data contribute to reinforce the postulation that physical activity supports healthy aging, particularly by helping to protect the immunological system from aging-related changes.Xunta de Galicia; ED431B 2016/013Xunta de Galicia; GPC2014/08

Comparative Survival Analysis of Deinococcus Radiodurans and the Haloarchaea Natrialba Magadii and Haloferax Volcanii, Exposed to Vacuum Ultraviolet Irradiation

The haloarchaea Natrialba magadii and Haloferax volcanii, as well as the radiation-resistant bacterium Deinococcus radiodurans, were exposed to vacuum-UV (V-UV) radiation at the Brazilian Synchrotron Light Laboratory (LNLS). Cell monolayers (containing 105 - 106 cells per sample) were prepared over polycarbonate filters and irradiated under high vacuum (10-5 Pa) with polychromatic synchrotron radiation. N. magadii was remarkably resistant to high vacuum with a survival fraction of ((3.77 \pm 0.76) x 10-2), larger than the one of D. radiodurans ((1.13 \pm 0.23) x 10-2). The survival fraction of the haloarchaea H. volcanii, of ((3.60 \pm 1.80) x 10-4), was much smaller. Radiation resistance profiles were similar between the haloarchaea and D. radiodurans for fluencies up to 150 J m-2. For fluencies larger than 150 J m-2 there was a significant decrease in the survival of haloarchaea, and in particular H. volcanii did not survive. Survival for D. radiodurans was 1% after exposure to the higher V-UV fluency (1350 J m-2) while N. magadii had a survival lower than 0.1%. Such survival fractions are discussed regarding the possibility of interplanetary transfer of viable micro-organisms and the possible existence of microbial life in extraterrestrial salty environments such as the planet Mars and the Jupiter's moon Europa. This is the first work reporting survival of haloarchaea under simulated interplanetary conditions.Comment: Draft version (without figures), Accepted for publication in Astrobiolog

Bioremediation aims at using organisms to solve environmental problems such as soil and groundwater contamination

Podemos definir biorremediación como la utilización de seres vivos para solucionar un problema ambiental, tales como suelo o agua subterránea contaminados. En un ambiente no contaminado, las bacterias, los hongos, los protistas, y otros microorganismos heterotróficos degradan constantemente la materia orgánica disponible, para obtener energía. Cuando un agente contaminante orgánico, combustible, petróleo u otro es accidentalmente liberado en un ambiente dado, algunos de los microorganismos indígenas morirán, mientras que sobrevivirían algunos otros capaces de degradar estos compuestos orgánicos. La biorremediación trabaja proveyendo a estos organismos de nutrientes, oxígeno, y otras condiciones que favorezcan su rápido crecimiento y reproducción. Estos organismos entonces podrán degradar el agente contaminante orgánico a una velocidad mayor, proporcionando una técnica para limpiar la contaminación, realzando los mismos procesos de biodegradación que ocurren naturalmente en el medio ambiente. Dependiendo del sitio y de sus contaminantes, la biorremediación puede ser más segura y menos costosa que soluciones alternativas tales como la incineración o el enterramiento de los materiales contaminados.In a non-polluted environment, bacteria, fungi, protists, and other heterotrophic microorganisms are constantly degrading organic matter to obtain energy. When organic pollutants such as fuel or oil accidentally spill, some of the indigenous microorganisms may die, while those capable of degrading the fuel may survive. Bioremediation enhances the activity of the later by providing fertilizers, oxygen, and by creating optimum conditions for rapid growth. These organisms may then be able to degrade the organic pollutant at a faster rate. Cleaning up pollution is thus performed by enhancing natural biodegradation processes. Depending on the site and the contaminants, bioremediation may be safer and less expensive than alternative solutions such as incineration and landfilling of contaminated materials

Isolation and Characterization of a Novel Electrogenic Bacterium, Dietzia sp. RNV-4.

Electrogenic bacteria are organisms that can transfer electrons to extracellular electron acceptors and have the potential to be used in devices such as bioelectrochemical systems (BES). In this study, Dietzia sp. RNV-4 bacterium has been isolated and identified based on its biochemical, physiological and morphological characteristics, as well as by its 16S rRNA sequence analysis. Furthermore, the current density production and electron transfer mechanisms were investigated using bioelectrochemical methods. The chronoamperometric data showed that the biofilm of Dietzia sp. RNV-4 grew as the current increased with time, reaching a maximum of 176.6 ± 66.1 mA/m2 at the end of the experiment (7 d); this highly suggests that the current was generated by the biofilm. The main electron transfer mechanism, indicated by the cyclic voltammograms, was due to secreted redox mediators. By high performance liquid chromatography, canthaxanthin was identified as the main compound involved in charge transfer between the bacteria and the solid electrodes. Dietzia sp. RNV-4 was used as biological material in a microbial fuel cell (MFC) and the current density production was 299.4 ± 40.2 mA/m2. This is the first time that Dietzia sp. RNV-4 has been electrochemically characterized and identified as a new electrogenic strain

Hydrophobic Forces Are Relevant to Bacteria-Nanoparticle Interactions: Pseudomonas putida Capture Efficiency by Using Arginine, Cysteine or Oxalate Wrapped Magnetic Nanoparticles

Size, shape and surface characteristics strongly affect interfacial interactions, as the presented among iron oxide nanoparticles (NPs) aqueous colloids and bacteria. In other to find the forces among this interaction, we compare three types of surface modified NPs (exposing oxalate, arginine or cysteine residues), based on a simple synthesis and derivation procedure, that allows us to obtain very similar NPs (size and shape of the magnetic core). In this way, we assure that the main difference in the synthesized NPs are the oxalate or amino acid residue exposed, an ideal situation to compare their bacterial capture performance, and so too the interactions among them. Field emission scanning electron microscopy showed homogeneous distribution of particle sizes for all systems synthesized, close to 10 nm. Magnetization, zeta potential, Fourier transformed infrared spectrometry and other studies allow us further characterization. Capture experiments of Pseudomonas putida bacterial strain showed a high level of efficiency, independently of the amino acid used to wrap the NP, when compared with oxalate. We show that bacterial capture efficiency cannot be related mostly to the bacterial and NP superficial charge relationship (as determined by z potential), but instead capture can be correlated with hydrophobic and hydrophilic forces among them