Los autores agradecen a la Oficina de Investigación Naval, Junta Unidas de soja, y la Fundación Nacional de Ciencias para la financiación.

1. Modificación de Nafion con sales de amonio cuaternario <ol><li> Agitar una botella de suspensión Nafion 5% w / v vigorosamente durante aprox. 30 segundos para asegurar que Nafion está suspendida de manera uniforme en la solución. </li><li> Pipetear a cabo 2 ml de la Nafion ahora re-suspendido en un vial de vidrio (volumen vial podría contener de 2,5 ml a 10 ml). </li><li> Medir un exceso molar de 3 veces (en relación con los grupos ácidos sulfónicos en el polímero Nafion) de la sal de amonio bromuro de alquilo (masas apropiadas se muestran en la Tabla 1) y añadir esta al vial que contiene el 2 ml de Nafion. </li><li> Vórtice del vial a 1500 rpm durante 10-15 minutos. </li><li> Vierta la solución viscosa en una bandeja de plástico un peso que mide aprox. 3 x 3 cm, y utilizar una pipeta para transferir cualquier solución residual del vial a la bandeja de pesaje. </li><li> Permitir los disolventes que se evapore de la barca pesan, dejando un color amarillo / marrón, película transparente en la parte inferior de la bandeja de pesaje (FiFigura 2). La tasa de evaporación del disolvente debe ser tal que se necesita más de 6 horas durante todo el disolvente se evapore. Si el solvente se evapora demasiado rápido, un material blanco, crujiente formará en lugar de una película transparente que indica que la estructura micelar del polímero ha sido destruido, y debe volver a iniciar el procedimiento. Si la evaporación del disolvente es demasiado lento, un deshumidificador puede ser necesario, debido a la evaporación demasiado lenta por lo general conduce a un gel pegajoso, de color naranja y debe volver a iniciar el procedimiento. Los rangos típicos de temperatura para la evaporación del disolvente son 20 a 37 ° C. Las condiciones reales para el secado son una función de la humedad relativa y temperatura de la habitación, pero es importante que el secado es lento para mantener la estructura micelar, pero no demasiado lento para permitir la formación de gel. </li><li> Llenar el bote con un peso 18M Ωcm agua desionizada (10-20 ml de agua), tapar y dejar en remojo durante 12-24 horas para eliminar el exceso de sales de bromuro de alquil amonio y HBr. </li><li&gt; Vierta (o pipeta) el agua y enjuague 3 veces con agua desionizada suficiente para llenar la bandeja cada vez. Tenga cuidado de no perder ninguna de la película de polímero durante este paso. </li><li> Permitir la bandeja de pesaje para sentarse cubierto hasta que el polímero está completamente seco En este punto, el polímero debe ser una película de plástico transparente y quebradizo algo. De nuevo, si el aire es muy húmedo, un deshumidificador puede ser necesario para completar la evaporación de una manera oportuna. </li><li> Usando una espátula, retire con cuidado la película seca de la bandeja de pesaje y la transfiere en un frasco de vidrio limpio. </li><li> Añadir 2 ml de etanol y 3 perlas de mezcla de cerámica, y vórtice durante 4 horas o hasta que la película de polímero está completamente re-suspendido. </li></ol> 2. La inmovilización de las enzimas en TBAB-Modified Nafion de Ensayos de actividad <ol><li> Para una enzima seca, pésese 1-10 mg de la enzima en un tubo de microcentrífuga de 1,5 ml, y se añade 1 ml de 100 mM de tampón fosfato de pH 7 para crear un 1-10 mg / ml de solución enzimática. For una enzima que está en solución, utilizar un ácido bicinconínico (BCA) ensayo 13 para determinar la cantidad de proteína, y añadir una cantidad apropiada de tampón fosfato 100 mM a reducir la concentración de proteína a 1-10 mg / ml. Ml 1-10 mg / normalmente corresponde a 1-50 nanomoles por ml. </li><li> Para 120 l de la de 1 mg / ml de solución enzimática, añadir 60 l de la solución Nafion alquilo amonio-modificado, y vórtice durante 10 segundos. (Esta mezcla puede ser ampliado para un gran número de repeticiones. Mantener la relación de solución de enzima a polímero a 2:1.) </li><li> Pipeta 60 l de la solución de enzima / polímero en la parte inferior de 3 separados 1 cm 2 cubetas, y dejar secar durante la noche. </li></ol> 3. Ensayo de la enzima inmovilizada deshidrogenasa NAD-dependiente <ol><li> Para la cubeta, añadir 1,3 ml de 50 mM de pirofosfato de sodio (pH 8,8), 1,5 ml de 15 mM de NAD (recién preparada) y 0,1 ml de agua. </li><li> Coloque la cubeta en un espectrofotómetro UV / Vis (Evolut ThermoScientific es decir,iones de Bio 260 y Thermo Spectronic Genesys 20) establece en una longitud de onda de 340 nm. </li><li> Poner a cero el espectrómetro y, a continuación, añadir 0,1 ml de etanol. Mezcle los reactivos pipeteando suavemente la solución arriba y hacia abajo 5 veces. Para un blanco, utilizar 0,1 ml de agua adicional en lugar de la 0,1 ml de etanol. </li><li> Anotar la absorbancia a 340 nm a 5 minutos después de que los reactivos se añadieron a la cubeta y 20 minutos después. Trazar los dos puntos de datos para conseguir una pendiente que puede ser utilizado para los cálculos de actividad. </li></ol> 4. Ensayo de inmovilizados PQQ-deshidrogenasas dependientes <ol><li> Para la cubeta, añadir 1,5 ml de fosfato sódico (pH 7,3) y 200 l de 600 uM PMS. </li><li> Colocar la cubeta en un espectrofotómetro UV / Vis establece en una longitud de onda de 600 nm y luego ajustar el cero del espectrómetro. </li><li> Añadir 100 l de 700 l DCIP y 200 l de sustrato de interés (etanol, acetaldehído, glicerol, glucosa, o gliceraldehído), y mezclar los reactivos pipeteando suavemente la solutión arriba y hacia abajo 5 veces. Para un blanco, utilice 200 l de agua en lugar del substrato de interés. </li><li> Anotar la absorbancia a 600 nm a 5 minutos después de que los reactivos se añadieron a la cubeta y 20 minutos después. </li></ol> 5. Ensayo de glucosa oxidasa inmovilizada <ol><li> Para la cubeta, añadir 2,0 ml de una solución que contiene 0,2 M ácido p-hidroxibenzoico, 0,02% (w / v) de azida de sodio, 128 U de peroxidasa, 0,3 mM de 4-aminoantipirina, 1 M de fosfato de potasio, y 50 mM de glucosa. Mezclar la solución con la pipeta hacia arriba y abajo 5 veces. </li><li> Colocar la cubeta en un conjunto espectrofotómetro UV / Vis a una longitud de onda de 510 nm. </li><li> Leer la absorbancia a 510 nm a 5 minutos después de que los reactivos se añadieron a la cubeta y de nuevo a 20 minutos después. </li></ol> 6. Los resultados representativos La estructura micelar del polímero Nafion modificado puede ser interrumpido por el secado de la sal original / polímero fundido co-película también fast. La figura 2 muestra una mezcla de sal / polímero que se ha secado correctamente resultante en una película transparente, de color marrón claro. Una película que se seca demasiado rápido puede resultar en escamas blancas opacas, de polímero debido al hecho de que el proceso de secado puede destruir la estructura micelar. Una vez que la modificación Nafion polímero y la enzima se han mezclado y co-vierte sobre el fondo de una cubeta, los ensayos de actividad enzimática se puede utilizar para evaluar la estabilidad de la enzima dentro de la película de polímero. Tablas 2-4 muestran los resultados del ensayo de dos enzimas deshidrogenasa y glucosa oxidasa inmovilizada en varias películas Nafion modificados, respectivamente. Nótese la mayor actividad de las enzimas que están inmovilizadas vs las enzimas en solución tampón, que muestra que los polímeros modificados Nafion puede realmente aumentar la actividad de ciertas enzimas (llamado superactivity). Otras enzimas tienen limitaciones de transporte que disminuyen su actividad específica, cuando inmovilizarlos en el polímero (es decir, celulasas y amilasas, cuyos sustratos son macromoléculas bastante grande). <table border="1"><tbody><tr><td> Sal de amonio cuaternario usado </td><td> 3 veces el exceso </td></tr><tr><td> T3A (bromuro de tetrapropilamonio) </td><td> 32,37 mg / ml </td></tr><tr><td> TBAB (bromuro de tetrabutilamonio) </td><td> 39,19 mg / ml </td></tr><tr><td> TPAB (bromuro de tetrapentilamonio) </td><td> 46,01 mg / ml </td></tr><tr><td> TEHA (bromuro de triethylhexylammonium) </td><td> 32,37 mg / ml </td></tr><tr><td> TMHA (bromuro de trimethylhexylammonium) </td><td> 27,25 mg / ml </td></tr><tr><td> TMOA (bromuro de trimethyloctylammonium) </td><td> 30,66 mg / ml </td></tr><tr><td> TMDA (bromuro de trimethyldecylammonium) </td><td> 34,07 mg / ml </td></tr><tr><td> TMDDA (bromuro de trimethyldodecylammonium) </td><td> 37,48 mg / ml </td></tr><tr><td> TMTDA (bromuro de trimethyltetradecylammonium) </td><td&gt; 40,89 mg / ml </td></tr><tr><td> TMHDA (bromuro de trimethylhexadecylammonium) </td><td> 44,31 mg / ml </td></tr><tr><td> TMODA (bromuro de trimethyloctadecylammonium) </td><td> 47,71 mg / ml </td></tr></tbody></table> Tabla 1. Las cantidades de sales de amonio tetra-alquil utilizar para Nafion modificación de polímeros. <table border="1"><tbody><tr><td> Tipo de Nafion </td><td> La actividad enzimática (U / g) </td></tr><tr><td> Tampón (sin polímero) </td><td> 16,63 ± 8,11 </td></tr><tr><td> Nafion (un-mod.) </td><td> 9,25 ± 2,21 </td></tr><tr><td> TMTDA </td><td> 3,23 ± 2,92 </td></tr><tr><td> TBAB </td><td> 3,93 ± 3,33 </td></tr><tr><td> TMDDA </td><td> 4,19 ± 1,04 </td></tr><tr><td> TMOA </td><td> 3,51 ± 1,11 </td></tr><tr><td> TMDA </td><td> 8,00 ± 4,53 </td></tr><tr><td> TMHA </td><td> 1,68 ± 1,39 </td></tr><tr><td> TMHDA </td><td> 4,83 ± 0,99 </td></tr><tr><td> TMODA </td><td> 10,45 ± 3,20 </td></tr></tbody></table> Tabla 2 NAD-dependiente de la actividad deshidrogenasa de glucosa inmovilizada en determinados polímeros modificados Nafion (nota: actividad inmovilizada es una función de la actividad inicial específica de la enzima).. <table border="1"><tbody><tr><td> Tipo de Nafion </td><td> La actividad enzimática (mU / g) </td></tr><tr><td> Tampón (sin polímero) </td><td> 7,18 ± 0,51 </td></tr><tr><td> Nafion (un-mod.) </td><td> 70,1 ± 0,5 </td></tr><tr><td> TMTDA </td><td> 133 ± 6 </td></tr><tr><td> TBAB </td><td> 244 ± 4 </td></tr><tr><td> TMDDA </td><td> 221 ± 6 </td></tr><tr><td> TMOA </td><td> 1,78 ± 0,63 </td></tr><tr><td> TMDA </td><td> 206 ±5 </td></tr><tr><td> TEHA </td><td> 40,1 ± 50,6 </td></tr><tr><td> TMHDA </td><td> 0 </td></tr><tr><td> TMODA </td><td> 1,45 ± 0,06 </td></tr></tbody></table> Tabla 3 PQQ-dependiente de la actividad deshidrogenasa de glucosa inmovilizada en determinados polímeros modificados Nafion (nota: actividad inmovilizada es una función de la actividad inicial específica de la enzima).. <table border="1"><tbody><tr><td> Tipo de Nafion </td><td> La actividad enzimática (U / g) </td></tr><tr><td> Tampón (sin polímero) </td><td> 103,61 ± 3,15 </td></tr><tr><td> Nafion (un-mod.) </td><td> 19,93 ± 10,10 </td></tr><tr><td> TMTDA </td><td> 247,25 ± 12,49 </td></tr><tr><td> TBAB </td><td> 152,27 ± 5,29 </td></tr><tr><td> TMDDA </td><td> 262,05 ± 6,26 </td></tr><tr><td> TMOA </td><td> 129,18 ± 2,31 </td></tr&gt; <tr><td> TMDA </td><td> 141,23 ± 1,97 </td></tr><tr><td> TMHA </td><td> 131,75 ± 2,89 </td></tr><tr><td> TMHDA </td><td> 132,50 ± 1,18 </td></tr><tr><td> TMODA </td><td> 136,50 ± 0,96 </td></tr></tbody></table> . Representante Tabla 4 glucosa oxidasa actividad específica inmovilizado en determinados polímeros modificados Nafion (nota: actividad inmovilizada es una función de la actividad inicial específica de la enzima). <img alt="Figura 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> Figura 1. Esquema de incorporación TBAB en Nafion polímero y su uso posterior en la inmovilización de enzimas. <img alt="Figura 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> Figura 2. Fotografía óptica de un primer reparto de co-películas de Nafion y TBAB. Reduzca la velocidad de secado se obtiene un film transparente, de color marrón claro que cubre el bottom de la bandeja de pesaje.

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No hay conflictos de interés declarado.

En el procedimiento descrito, tetra-alquil sales de amonio se utilizan para modificar Nafion comercial para crear polímeros micelares que se pueden utilizar para inmovilizar y estabilizar las enzimas. Los ensayos descritos en el procedimiento muestran que el polímero puede ser usado para inmovilizar una amplia variedad de enzimas con una alta retención de la actividad. Si la enzima de interés tiene una actividad muy baja o es impura, una mayor concentración puede ser necesario y no debe afectar el proceso de inmovi...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Nafion </td><td> Sigma-Aldrich </td><td> 70160 </td></tr><tr><td> Tetra sales de bromuro de alquilamonio </td><td> Sigma-Aldrich </td><td> n / a </td></tr><tr><td> El alcohol deshidrogenasa </td><td> Sigma-Aldrich </td><td> A3263 </td></tr><tr><td> Dinucleótido de nicotinamida adenina (NAD) </td><td> SIGMA-Aldrich </td><td> N7004 </td></tr><tr><td> Sodio pirofosfato </td><td> Sigma-Aldrich </td><td> P8010 </td></tr><tr><td> Fenazina metosulfato (PMS) </td><td> Sigma-Aldrich </td><td> P9625 </td></tr><tr><td> 2,6-Dichloroindophenol (DCIP) </td><td> Sigma-Aldrich </td><td> D1878 </td></tr><tr><td> La glucosa oxidasa </td><td> Sigma-Aldrich </td><td> G7141 </td></tr><tr><td> Ácido 4-hidroxibenzoico </td><td> Sigma-Aldrich </td><td> 240141 </td></tr><tr><td> Azida de sodio </td><td> Sigma-Aldrich </td><td> S8032 </td></tr><tr><td> Peroxidasa </td><td> Sigma-Aldrich </td><td> P8375 </td></tr><tr><td> 4-aminoantipirina </td><td> Sigma-Aldrich </td><td> 06800 </td></tr><tr><td> Espectrofotómetro UV / VIS </td><td> Thermo </td><td> Evolución 260 Bio o Spectronic Genesys 20 </td></tr><tr><td> Vortex Genie </td><td></td><td></td></tr><tr><td> Balanza analítica </td><td></td><td></td></tr></tbody></table&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Durante la última década, ha habido una gran cantidad de solicitud de enzimas inmovilizadas y estabilizado incluyendo biocatálisis, biosensores, y las células de biocombustible. 1-3 En aplicaciones más bioelectrochemical, enzimas u orgánulos se inmovilizan sobre una superficie del electrodo con el uso de algún tipo de polímero de matriz. Este andamio polímero debe mantener las enzimas estable y permitir la difusión superficial de las moléculas y los iones en y fuera de la matriz. La mayoría de los polímeros utilizados para este tipo de inmovilización se basan en poliaminas o polialcoholes - polímeros que imitan el entorno natural de las enzimas que se encapsulan y estabilizar la enzima a través de hidrógeno o un enlace iónico. Otro método para estabilizar las enzimas implica el uso de micelas, que contienen regiones hidrofóbicas que pueden encapsular y estabilizar las enzimas 4,5. En particular, el grupo Minteer ha desarrollado un polímero basado en micelar Nafion comercialmente disponible. 6,7 Nafionen sí es un polímero micelar que permite la difusión del canal con ayuda de protones y otros cationes pequeños, pero las micelas y los canales son extremadamente pequeñas y el polímero es muy ácida debido a las cadenas laterales de ácido sulfónico, que es desfavorable para la inmovilización de enzimas. Sin embargo, cuando Nafion se mezcla con un exceso de sales de amonio hidrófobos alquilo tales como bromuro de tetrabutilamonio (BTBA), los cationes de amonio cuaternario reemplazar los protones y se convierten en los contra-iones de los grupos sulfonato en las cadenas laterales del polímero (Figura 1). Esto se traduce en mayores micelas y canales dentro del polímero que permiten la difusión de sustratos grandes e iones que son necesarios para la función enzimática, tales como nicotinamida adenina dinucleótido (NAD). Esta modificación Nafion polímero se ha utilizado para inmovilizar muchos tipos diferentes de enzimas, así como las mitocondrias para su uso en biosensores y células de combustible biológico. 8-12 Este artículo describe un nuevo procedimiento para la fabricación de este micrófonoEllar enzima polímero inmovilización membrana que puede estabilizar las enzimas. La síntesis de la membrana de inmovilización de enzimas micelar, el procedimiento para inmovilizar enzimas dentro de la membrana, y los ensayos para estudiar la actividad enzimática específica de la enzima inmovilizada se detallan a continuación.

Watch this Scientific Journal Video about Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization at JoVE.com

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

En este artículo se describe el procedimiento para la síntesis de una enzima modificada hidrófobamente membrana Nafion inmovilización y cómo inmovilizar las proteínas y / o enzimas dentro de la membrana y probar su actividad específica.

Hidrófobo sal modificada Nafion para inmovilización de enzimas y Estabilización

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

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16.1K vistas.  University of Utah. En este artículo se describe el procedimiento para la síntesis de una enzima modificada hidrófobamente membrana Nafion inmovilización y cómo inmovilizar las proteínas y / o enzimas dentro de la membrana y probar su actividad específica.

Video: Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Inmovilización de bacterias para tratamiento de imágenes de microscopía de fuerza atómica

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Investigación

Bioingeniería

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: una plataforma automatizada para el descubrimiento de enzimas de la biomasa y la optimización Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Capa por capa deposición de colágeno en dispositivos de microfluidos para microtejidos Estabilización

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Química húmeda y el péptido Inmovilización de politetrafluoroetileno para la adhesión celular mejorada

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted membrana plasmática Entrega de cargo encapsulado hidrofóbico en un cristal líquido portador de nanopartículas

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Dirigida la inmovilización de la proteína ósea morfogenética 2 a superficies sólidas por química del tecleo

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Fabricación de una cubierta de Nafion, reduce el óxido/polianilina Chemiresistive Sensor de pH Monitor en tiempo real durante la fermentación microbiana

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Superficie-Inmovilización funcional de Genes utilizando litografía de desplazamiento de varias fases de la cadena

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

Flash NanoPrecipitation para la encapsulación de compuestos hidrofóbicos e hidrofílicos en nanopartículas poliméricas

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Inmovilización de personas en Caenorhabditis elegans con un Chip de microfluidos polidimetilsiloxano ultra-delgada de retención de agua

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...