Les auteurs tiennent à remercier l&#39;Office of Naval Research, United Soybean Board, et la National Science Foundation pour le financement.

1. Modification de Nafion avec sels d&#39;ammonium quaternaire <ol><li> Agiter une bouteille de suspension Nafion 5% p / v vigoureusement pendant environ. 30 secondes pour s&#39;assurer que le Nafion est suspendu de manière uniforme dans la solution. </li><li> Introduire à la pipette sur 2 mL de la Nafion désormais remis en suspension dans un flacon en verre (volume du flacon peut contenir de 2,5 ml à 10 ml). </li><li> Mesurer un excès de 3 fois molaire (par rapport à des groupes acide sulfonique sur le polymère Nafion) du sel de bromure d&#39;ammonium alkyle (masses appropriées sont indiquées dans le tableau 1) et l&#39;ajouter à la fiole contenant le 2 mL de Nafion. </li><li> Vortex du flacon à 1500 rpm pendant 10-15 minutes. </li><li> Verser la solution visqueuse dans un bac en plastique de pesage qui mesure environ. 3 x 3 po, et d&#39;utiliser une pipette pour transférer toute solution résiduelle de la fiole sur le plateau de pesée. </li><li> Permettre aux solvants pour évaporer du bateau poids, en laissant un jaune / brun, un film transparent sur ​​le fond du plateau de pesage (Figure 2). Le taux d&#39;évaporation des solvants devrait être telle qu&#39;elle prend plus de 6 heures pour tout le solvant s&#39;évapore. Si le solvant s&#39;évapore trop vite, un matériau blanc croustillant formeront, au lieu d&#39;un film transparent qui indique que la structure micellaire du polymère a été détruit, et vous devez redémarrer la procédure. Si l&#39;évaporation du solvant est trop lent, un déshumidificateur peut être nécessaire, parce que trop lente de l&#39;évaporation conduit généralement à un collant, un gel orange et vous devez redémarrer la procédure. Plages de température typiques pour l&#39;évaporation du solvant sont de 20 à 37 ° C. Les conditions réelles pour le séchage sont fonction de l&#39;humidité relative et la température dans la chambre, mais il est important que le séchage est lent pour maintenir la structure des micelles, mais pas trop lent pour permettre la formation de gel. </li><li> Remplissez la pesée bateau avec 18M Qcm de l&#39;eau déminéralisée (10-20 ml d&#39;eau), la couverture, et laisser tremper pendant 12-24 heures pour éliminer l&#39;excès de sels d&#39;alkyl ammonium bromure et HBr. </li><li&gt; Versez (ou une pipette) à l&#39;eau et rincer 3 fois avec DI assez d&#39;eau pour remplir le bac à chaque fois. Veillez à ne pas perdre de la pellicule de polymère au cours de cette étape. </li><li> Laissez le plateau de pesée de s&#39;asseoir à découvert jusqu&#39;à ce que le polymère est complètement sec À ce stade, le polymère doit être un film clair et un peu cassant en plastique. Encore une fois, si l&#39;air est très humide, un déshumidificateur peut être nécessaire pour compléter l&#39;évaporation dans les meilleurs délais. </li><li> Avec une spatule, retirez délicatement le film séché à partir du bac de pesage et de le transférer dans un flacon en verre propre. </li><li> Ajouter 2 ml d&#39;éthanol et 3 perles en céramique de mélange, et le vortex pendant 4 heures ou jusqu&#39;à ce que le film de polymère est complètement remis en suspension. </li></ol> 2. Immobilisation d&#39;enzymes dans TBAB-mise à jour pour le Nafion tests d&#39;activité <ol><li> Pour une enzyme sec, peser 1-10 mg de l&#39;enzyme dans un microtube de 1,5 mL, et ajouter 1 mL de 100 mM de tampon phosphate pH 7 pour créer un 1-10 mg / ml solution enzymatique. For une enzyme qui est en solution, utiliser un acide bicinchoninique (BCA) test 13 pour déterminer la quantité de protéines, et ajouter une quantité appropriée de tampon phosphate 100 mM pour amener la concentration de protéines à 1-10 mg / ml. 1-10 ml mg / correspond typiquement à 1-50 nanomoles / mL. </li><li> À 120 pi de 1 mg / ml solution enzymatique, ajouter 60 ul de la solution de Nafion d&#39;ammonium modifié alkyle, et vortex pendant 10 secondes. (Ce mélange peut être étendu à un grand nombre de répétitions. Garder le rapport solution enzymatique à polymère à 2:1.) </li><li> Introduire à la pipette 60 pi de la solution enzymatique / polymère dans le bas de 3 séparées de 1 cm 2 cuves, et laisser sécher pendant la nuit. </li></ol> 3. Le dosage de l&#39;enzyme immobilisée déshydrogénase NAD-dépendante <ol><li> Pour la cuvette, ajoutez 1,3 ml de 50 mM de pyrophosphate de sodium (pH 8,8), 1,5 ml de 15 mM NAD (fraîchement préparée), et 0,1 mL d&#39;eau. </li><li> Placer la cuvette dans un spectrophotomètre UV / VIS (c.-à-Evolut ThermoScientificion 260 Bio et Thermo Spectronic Genesys 20) fixée à une longueur d&#39;onde de 340 nm. </li><li> Zéro du spectromètre, puis ajouter 0,1 ml d&#39;éthanol. Mélanger les réactifs par pipetage doucement la solution de haut en bas 5 fois. Pour un blanc, utiliser 0,1 ml d&#39;eau supplémentaire au lieu de la 0,1 mL d&#39;éthanol. </li><li> Enregistrez l&#39;absorbance à 340 nm à 5 minutes après les réactifs ont été ajoutés à la cuvette et 20 minutes après. Tracer les deux points de données pour obtenir une pente qui peut être utilisé pour le calcul d&#39;activité. </li></ol> 4. Dosage de la immobilisées PQQ-dépendants déshydrogénases <ol><li> Pour la cuvette, ajoutez 1,5 ml de phosphate de sodium (pH 7,3) et 200 ul de 600 uM PMS. </li><li> Placer la cuvette dans un spectrophotomètre UV / Vis réglé à une longueur d&#39;onde de 600 nm, puis zéro le spectromètre. </li><li> Ajouter 100 ul de 700 DCIP ul et 200 ul du substrat d&#39;intérêt (l&#39;éthanol, l&#39;acétaldéhyde, le glycérol, le glucose, ou de glycéraldéhyde), et mélanger les réactifs par un léger pipetage la solutions de haut en bas 5 fois. Pour un blanc, utiliser 200 pi d&#39;eau au lieu du substrat d&#39;intérêt. </li><li> Enregistrez l&#39;absorbance à 600 nm à 5 minutes après les réactifs ont été ajoutés à la cuvette et 20 minutes après. </li></ol> 5. Dosage de la glucose-oxydase immobilisée <ol><li> Ensemble de la cuvette, ajouter 2,0 ml d&#39;une solution contenant 0,2 M d&#39;acide p-hydroxybenzoïque, l&#39;azoture de sodium 0,02% (p / v), la peroxydase 128 U, 0,3 mM de 4-aminoantipyrine, une M de phosphate de potassium, et 50 mM de glucose. Mélanger la solution par aspiration et refoulement 5 fois. </li><li> Placer la cuvette dans un ensemble spectrophotomètre UV / Vis à une longueur d&#39;onde de 510 nm. </li><li> Enregistrez l&#39;absorbance à 510 nm à 5 minutes après les réactifs ont été ajoutés à la cuvette et encore moins 20 minutes après. </li></ol> 6. Les résultats représentatifs La structure micellaire du polymère Nafion modifié peut être perturbé par le séchage du sel d&#39;origine / polymère co-moulé film trop fast. La figure 2 montre un mélange sel / polymère qui a été séché correctement résultant d&#39;une manière transparente, brun léger film. Un film qui sèche trop rapide peut entraîner opaques, de flocons de polymère blanc en raison du fait que le processus de séchage peut détruire la structure micellaire. Une fois que le Nafion polymère modifié et de l&#39;enzyme ont été mélangés et co-coulée sur le fond d&#39;une cuvette, les dosages d&#39;activité enzymatique peut être utilisé pour évaluer la stabilité de l&#39;enzyme dans le film polymère. Tableaux 2-4 montrent les résultats d&#39;analyse de deux enzymes déshydrogénases et la glucose oxydase immobilisée dans divers films de Nafion modifiés, respectivement. Notez la plus grande activité des enzymes qui sont immobilisées vs les enzymes en solution tampon, montrant que les polymères Nafion modifiés peuvent en fait accroître l&#39;activité de certaines enzymes (appelé suractivité). D&#39;autres enzymes ont des limitations de transport qui réduisent leur activité spécifique lorsque les immobiliser dans le polymère (cellulases et amylases, c.-à-dont les substrats sont assez grandes macromolécules). <table border="1"><tbody><tr><td> Sel d&#39;ammonium quaternaire utilisé </td><td> 3 fois plus </td></tr><tr><td> T3A (bromure de tétrapropylammonium) </td><td> 32,37 mg / ml </td></tr><tr><td> TBAB (bromure de tétrabutylammonium) </td><td> 39,19 mg / ml </td></tr><tr><td> TPAB (bromure de tétrapentylammonium) </td><td> 46,01 mg / ml </td></tr><tr><td> TEHA (bromure de triethylhexylammonium) </td><td> 32,37 mg / ml </td></tr><tr><td> TMHA (bromure de trimethylhexylammonium) </td><td> 27,25 mg / ml </td></tr><tr><td> TMOA (bromure de trimethyloctylammonium) </td><td> 30,66 mg / ml </td></tr><tr><td> TMDA (bromure de trimethyldecylammonium) </td><td> 34,07 mg / ml </td></tr><tr><td> TMDDA (bromure de trimethyldodecylammonium) </td><td> 37,48 mg / ml </td></tr><tr><td> TMTDA (bromure de trimethyltetradecylammonium) </td><td&gt; 40,89 mg / ml </td></tr><tr><td> TMHDA (bromure de triméthylhexadécylammonium) </td><td> 44,31 mg / ml </td></tr><tr><td> TMODA (bromure de trimethyloctadecylammonium) </td><td> 47,71 mg / ml </td></tr></tbody></table> Tableau 1. Montants de sels d&#39;ammonium tétra-alkyle à utiliser pour le Nafion modification de polymères. <table border="1"><tbody><tr><td> Type de Nafion </td><td> L&#39;activité enzymatique (U / g) </td></tr><tr><td> Tampon (aucun polymère) </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> Tableau 2 NAD-dépendante activité glucose déshydrogénase immobilisée dans sélectionnés polymères modifiés Nafion billet: activité immobilisée est une fonction de l&#39;activité initiale spécifique de l&#39;enzyme).. <table border="1"><tbody><tr><td> Type de Nafion </td><td> L&#39;activité enzymatique (mU / g) </td></tr><tr><td> Tampon (aucun polymère) </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> Tableau 3 PQQ-dépendante activité glucose déshydrogénase immobilisée dans sélectionnés polymères modifiés Nafion billet: activité immobilisée est une fonction de l&#39;activité initiale spécifique de l&#39;enzyme).. <table border="1"><tbody><tr><td> Type de Nafion </td><td> L&#39;activité enzymatique (U / g) </td></tr><tr><td> Tampon (aucun polymère) </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> . Représentant Tableau 4 glucose oxydase spécifique immobilisé dans certains polymères Nafion modifiés (note: l&#39;activité immobilisé est une fonction de l&#39;activité initiale spécifique de l&#39;enzyme). <img alt="Figure 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> Figure 1. Schéma de constitution TBAB en Nafion polymère et une utilisation ultérieure dans l&#39;immobilisation d&#39;enzymes. <img alt="Figure 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> Figure 2. Photographie optique d&#39;un premier co-casting des films de Nafion et TBAB. Séchage lent donne un film transparent, brun clair couvrant la boTTOM du plateau de pesage.

<ol>
	<li>Calabrese-Barton, S., Gallaway, J., Atanassov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+biofuel+cells+for+implantable+and+microscale+devices+Chem.">Enzymatic biofuel cells for implantable and microscale devices Chem.</a> Rev. 104, 4867-4886 (2004).</li><li>Cracknell, J. A., Vincent, K. A., Armstrong, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+as+Working+or+Inspirational+Electrocatalysts+for+Fuel+Cells+and+Electrolysis.">Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis.</a> Chem. Rev. 108, 2439-2461 (2008).</li><li>Minteer, S. D., Liaw, B. Y., Cooney, M. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Enzyme+Cascades+for+Methanol/Air+Biofuel+Cells+Based+On+NAD+-Dependent+Enzymes.">Evaluating Enzyme Cascades for Methanol/Air Biofuel Cells Based On NAD+-Dependent Enzymes.</a> Electroanalysis. 22, 807-812 (2010).</li><li>Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150, 76-85 (1985).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Dans le mode opératoire décrit, les sels d&#39;ammonium tétra-alkyle sont utilisés pour modifier le Nafion commerciale pour créer des polymères micellaires qui peuvent être utilisés pour immobiliser et stabiliser les enzymes. Les tests décrits dans le spectacle procédure que le polymère peut être utilisé pour immobiliser une grande variété d&#39;enzymes avec une forte rétention de l&#39;activité. Si l&#39;enzyme d&#39;intérêt a une activité très faible ou qui est impur, une concentration plus élev?...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td></tr><tr><td> Nafion </td><td> Sigma-Aldrich </td><td> 70160 </td></tr><tr><td> Sels de bromure de Tetra alkylammonium </td><td> Sigma-Aldrich </td><td> n / a </td></tr><tr><td> L&#39;alcool déshydrogénase </td><td> Sigma-Aldrich </td><td> A3263 </td></tr><tr><td> Nicotinamide adénine dinucléotide (NAD) </td><td> Simga-Aldrich </td><td> N7004 </td></tr><tr><td> Pyrophosphate de sodium </td><td> Sigma-Aldrich </td><td> P8010 </td></tr><tr><td> Méthosulfate de phénazine (PMS) </td><td> Sigma-Aldrich </td><td> P9625 </td></tr><tr><td> 2,6-dichloroindophénol (DCIP) </td><td> Sigma-Aldrich </td><td> D1878 </td></tr><tr><td> La glucose oxydase </td><td> Sigma-Aldrich </td><td> G7141 </td></tr><tr><td> L&#39;acide 4-hydroxybenzoïque </td><td> Sigma-Aldrich </td><td> 240141 </td></tr><tr><td> L&#39;azoture de sodium </td><td> Sigma-Aldrich </td><td> S8032 </td></tr><tr><td> Peroxydase </td><td> Sigma-Aldrich </td><td> P8375 </td></tr><tr><td> 4-aminoantipyrine </td><td> Sigma-Aldrich </td><td> 06800 </td></tr><tr><td> UV / Vis Spectrophotomètre </td><td> Thermo </td><td> Evolution 260 Bio ou Spectronic Genesys 20 </td></tr><tr><td> Vortex Genie </td><td></td><td></td></tr><tr><td> Balance analytique </td><td></td><td></td></tr></tbody></table&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Au cours de la dernière décennie, il a été une richesse d&#39;application pour les enzymes immobilisées et stabilisé, y compris la biocatalyse, les biocapteurs et les cellules de biocarburants. 1-3 Dans la plupart des applications bioélectrochimiques, des enzymes ou des organites sont immobilisés sur une surface de l&#39;électrode avec l&#39;utilisation d&#39;un certain type de matrice de polymère. Cet échafaudage polymère doit maintenir les enzymes stable et permettre la diffusion facile des molécules et des ions dans et hors de la matrice. La plupart des polymères utilisés pour ce type d&#39;immobilisation sont basées sur des polyamines ou des polyols - polymères qui imitent l&#39;environnement naturel des enzymes qu&#39;ils encapsuler et de stabiliser l&#39;enzyme par le biais d&#39;hydrogène ou une liaison ionique. Une autre méthode pour stabiliser les enzymes implique l&#39;utilisation de micelles, qui contiennent des régions hydrophobes qui peuvent encapsuler et de stabiliser les enzymes. 4,5 En particulier, le groupe a développé Minteer un polymère micellaire basée sur Nafion disponible dans le commerce. 6,7 Nafionlui-même est un polymère micellaire qui permet la diffusion de canal assistée de protons et d&#39;autres cations de petite taille, mais les micelles et les canaux sont extrêmement faibles et le polymère est très acide en raison de chaînes latérales d&#39;acides sulfoniques, ce qui est défavorable pour l&#39;immobilisation d&#39;enzymes. Cependant, lorsque le Nafion est mélangé avec un excès de sels d&#39;ammonium d&#39;alkyle hydrophobes tels que le bromure de tétrabutylammonium (TBAB), les cations ammonium quaternaire remplacer les protons et les contre-ions deviennent des groupes sulfonate sur les chaînes latérales polymères (figure 1). Il en résulte dans les grandes micelles et les canaux dans le polymère qui permettent la diffusion de substrats de grande taille et des ions qui sont nécessaires pour la fonction enzymatique de type nicotinamide adénine dinucléotide (NAD). Cette modification de Nafion polymère a été utilisé pour immobiliser de nombreux différents types d&#39;enzymes ainsi que des mitochondries pour une utilisation dans des biocapteurs et les cellules de biocarburants. 8-12 Cet article décrit une nouvelle procédure pour la fabrication de ce microellar membrane polymère immobilisation d&#39;enzymes qui peuvent stabiliser les enzymes. La synthèse de la membrane immobilisation d&#39;enzymes micellaire, la procédure d&#39;immobilisation d&#39;enzymes dans la membrane, et les essais pour étudier l&#39;activité enzymatique spécifique de l&#39;enzyme immobilisée sont détaillées ci-dessous.

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

Cet article décrit la procédure de synthèse d&#39;une membrane Nafion modifié hydrophobiquement enzyme immobilisation et comment immobiliser des protéines et / ou des enzymes au sein de la membrane et de tester leur activité spécifique.

Hydrophobe sel modifié Nafion pour l&#39;immobilisation d&#39;enzymes et de stabilisation

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 vues.  University of Utah. Cet article décrit la procédure de synthèse d'une membrane Nafion modifié hydrophobiquement enzyme immobilisation et comment immobiliser des protéines et / ou des enzymes au sein de la membrane et de tester leur activité spécifique.

Vidéo: 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).

Immobilisation bactérienne pour l&#39;imagerie par microscopie à force atomique

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 ...

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Bio-ingénierie

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: une plate-forme automatisée pour Discovery enzymatique de la biomasse et l&#39;optimisation 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.

Couche par couche dépôt de collagène dans les dispositifs microfluidiques pour Microbroyeur stabilisation

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.

Wet Chemistry and Peptide Immobilisation sur polytétrafluoroéthylène pour l&#39;amélioration de l&#39;adhérence cellulaire

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 Plasma Membrane Livraison d&#39;un Hydrophobe Cargo encapsulé dans un cristal liquide Nanoparticules Transporteur

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.

Immobilisation dirigée de protéine morphogénétique osseuse 2 aux Surfaces solides par Click Chemistry

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.

Fabrication d’un enduit Nafion, réduit le graphène oxyde/Polyaniline Chemiresistive capteur de pH de moniteur en temps réel au cours de la Fermentation microbienne

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.

Surface-immobilisation fonctionnelle des gènes à l’aide de la lithographie de déplacement multipas Strand

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 Nanoprécipitation pour l’Encapsulation des composés hydrophobes et hydrophiles en nanoparticules polymériques

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.

Immobilisation des individus vivants Caenorhabditis elegans en utilisant une puce de microfluidique de polydiméthylsiloxane ultra-mince avec rétention d’eau

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