Gli autori riconoscono l&#39;Office of Naval Research, consiglio di soia Uniti, e la National Science Foundation per il finanziamento.

1. Modifica del Nafion con sali di ammonio quaternario <ol><li> Agitare una bottiglia del 5% w / v sospensione Nafion vigorosamente per ca. 30 secondi per assicurare che Nafion viene sospeso in soluzione uniforme. </li><li> Pipettare in 2 mL di Nafion ora risospeso in una fiala di vetro (volume fiala può contenere da 2,5 ml a 10 ml). </li><li> Misura di 3 volte in eccesso molare (rispetto a gruppi di acido solfonico nel polimero Nafion) del sale di alchil ammonio bromuro (masse appropriate sono mostrati in Tabella 1) ed aggiungere al flaconcino contenente il 2 mL di Nafion. </li><li> Vortex il flacone a 1500 rpm per 10-15 minuti. </li><li> Versare la soluzione viscosa in un vassoio di plastica di peso che misura ca. 3 x 3 pollici, e utilizzare una pipetta per trasferire i residui della soluzione dal flaconcino al vassoio di pesatura. </li><li> Lasciare evaporare i solventi dal peso barca, lasciando un giallo / marrone, pellicola trasparente sul fondo del vassoio di pesatura (Figura 2). Il tasso di evaporazione del solvente deve essere tale che ci vuole più di 6 ore per evaporare tutto il solvente. Se il solvente evapora troppo velocemente, un bianco, croccante materiale formerà, invece di una pellicola trasparente che indica che la struttura micellare del polimero è stata distrutta, ed è necessario riavviare la procedura. Se evaporazione del solvente è troppo lento, un de-umidificatore può essere necessario, perché troppo lenta evaporazione porta normalmente a uno cattivo gusto, gel arancione ed è necessario riavviare la procedura. Intervalli di temperatura tipici per evaporazione del solvente sono 20 - 37 ° C. Le condizioni effettive di essiccazione sono funzione della temperatura e dell&#39;umidità relativa nella camera, ma è importante che l&#39;essiccazione essendo lento per mantenere struttura micellare, ma non troppo lento per permettere la formazione di gel. </li><li> Riempire il peso barca con 18M Qcm acqua deionizzata (10-20 ml di acqua), coprire e lasciare in immersione per 12-24 ore per rimuovere i sali in eccesso alchil ammonio bromuro e HBr. </li><li&gt; Versare (o pipetta) l&#39;acqua e lavare 3 volte con acqua deionizzata a sufficienza per riempire il cassetto ogni volta. Fare attenzione a non perdere uno dei film polimerico in questa fase. </li><li> Lasciare il vassoio di pesatura per sedersi scoperto fino a che il polimero è completamente asciutta A questo punto, il polimero dovrebbe essere una pellicola trasparente e piuttosto fragili plastica. Anche in questo caso, se l&#39;aria è molto umida, uno de-umidificatore può essere necessario per completare l&#39;evaporazione in modo tempestivo. </li><li> Utilizzando una spatola, rimuovere con cura la pellicola essiccata dal vassoio di pesatura e trasferirlo in una fiala di vetro pulito. </li><li> Aggiungere 2 mL di etanolo e 3 perle di ceramica di miscelazione e miscelare per 4 ore o fino a quando il film polimerico viene completamente ri-sospeso. </li></ol> 2. Immobilizzazione di enzimi in TBAB Nafion-Modified per saggi di attività <ol><li> Per un enzima secco, pesare 1-10 mg dell&#39;enzima in una provetta da 1,5 ml, e aggiungere 1 ml di 100 mM tampone fosfato pH 7 per creare un 1-10 mg / ml di soluzione enzimatica. For un enzima che è in soluzione, utilizzare un acido bicinconinico (BCA) dosaggio 13 per determinare la quantità di proteine, e aggiungere una quantità appropriata di 100 mM tampone fosfato per portare la concentrazione di proteina di 1-10 mg / mL. 1-10 mg mL / in genere corrisponde a 1-50 nanomoli / mL. </li><li> Per 120 microlitri della 1 mg / mL di soluzione enzimatica, aggiungere 60 ul di alchil ammonio-modificato soluzione di Nafion, e vortex per 10 secondi. (Questa miscela può essere scalato per un gran numero di repliche. Mantenere l&#39;enzima-to-polimero rapporto di soluzione 2:1.) </li><li> Pipettare 60 pl di dell&#39;enzima / polimero soluzione nel fondo 3 distinti 1 cm 2 cuvette, e lasciare asciugare durante la notte. </li></ol> 3. Saggio di immobilizzata NAD-Dependent Enzyme deidrogenasi <ol><li> Per la cuvetta, aggiungere 1,3 ml di 50 mM pirofosfato di sodio (pH 8,8), 1,5 ml di 15 mM NAD (preparati al momento), e 0,1 ml di acqua. </li><li> Posizionare la cuvetta in uno spettrofotometro UV / Vis (cioè Evolut ThermoScientificion 260 Bio e Thermo Spectronic Genesys 20) impostato su una lunghezza d&#39;onda di 340 nm. </li><li> Azzerare lo spettrometro, e quindi aggiungere 0,1 mL di etanolo. Mescolare i reagenti pipettando gentilmente la soluzione su e giù per 5 volte. Per un vuoto, usare 0,1 mL di acqua addizionale, invece del 0,1 mL di etanolo. </li><li> Registrare l&#39;assorbanza a 340 nm a 5 minuti dopo i reagenti sono stati aggiunti alla cuvetta e 20 minuti dopo. Tracciare i due punti di dati per ottenere una pendenza che può essere utilizzato per i calcoli di attività. </li></ol> 4. Saggio di immobilizzati PQQ-dipendenti deidrogenasi <ol><li> Per la cuvetta, aggiungere 1,5 ml di fosfato di sodio (pH 7,3) e 200 pl di 600 uM PMS. </li><li> Posizionare la cuvetta in uno spettrofotometro UV / Vis impostato a una lunghezza d&#39;onda di 600 nm e quindi azzerare il spettrometro. </li><li> Aggiungere 100 pl di 700 microlitri DCIP e 200 pl del substrato di interesse (etanolo, acetaldeide, glicerolo, glucosio, o gliceraldeide), e miscelare i reagenti pipettando gentilmente la soluzion su e giù per 5 volte. Per un vuoto, usare 200 pl di acqua invece del substrato di interesse. </li><li> Registrare l&#39;assorbanza a 600 nm a 5 minuti dopo i reagenti sono stati aggiunti alla cuvetta e 20 minuti dopo. </li></ol> 5. Saggio di glucosio ossidasi immobilizzato <ol><li> Per la cuvetta, aggiungere 2,0 ml di una soluzione contenente 0,2 M p-idrossibenzoico, sodio azide 0,02% (w / v), 128 U perossidasi, 0,3 mM di 4-amminoantipirina, 1 M di fosfato di potassio, e 50 mM di glucosio. Miscelare la soluzione pipettando su e giù 5 volte. </li><li> Posizionare la cuvetta in uno UV / Vis set spettrofotometro ad una lunghezza d&#39;onda di 510 nm. </li><li> Registrare l&#39;assorbanza a 510 nm a 5 minuti dopo i reagenti sono stati aggiunti alla cuvetta e di nuovo a 20 minuti dopo. </li></ol> 6. Risultati rappresentativi La struttura micellare del polimero Nafion modificata può essere interrotto con l&#39;essiccazione del sale originale / co-polimero fuso film troppo fast. Figura 2 mostra un sale / polimero miscela che è stata essiccata correttamente conseguente pellicola trasparente, marrone chiaro. Un film che asciuga troppo velocemente può provocare opachi, bianchi fiocchi di polimero a causa del fatto che il processo di asciugatura può distruggere la struttura micellare. Una volta che il polimero modificato Nafion e enzima sono stati miscelati e co-espressi sul fondo di una cuvetta, saggi di attività enzimatica può essere utilizzato per valutare la stabilità dell&#39;enzima all&#39;interno del film polimerico. Tabelle 2-4 mostrano i risultati del dosaggio dei due enzimi deidrogenasi e glucosio ossidasi immobilizzata in vari film Nafion modificati, rispettivamente. Nota la maggiore attività degli enzimi che sono immobilizzati contro gli enzimi in soluzione tampone, mostrando che i polimeri modificati Nafion può migliorare l&#39;attività di certi enzimi (chiamati superactivity). Altri enzimi hanno limitazioni di trasporto che riducono la loro attività specifica quando li immobilizzare il polimero (amilasi e cellulasi ad esempio, i cui substrati sono macromolecole piuttosto grandi). <table border="1"><tbody><tr><td> Sale di ammonio quaternario utilizzata </td><td> 3 volte più elevato </td></tr><tr><td> T3A (bromuro di tetrapropilammonio) </td><td> 32,37 mg / ml </td></tr><tr><td> TBAB (bromuro di tetrabutilammonio) </td><td> 39,19 mg / ml </td></tr><tr><td> TPAB (bromuro tetrapentilammonio) </td><td> 46,01 mg / ml </td></tr><tr><td> TEHA (bromuro triethylhexylammonium) </td><td> 32,37 mg / ml </td></tr><tr><td> TMHA (bromuro trimethylhexylammonium) </td><td> 27,25 mg / ml </td></tr><tr><td> TMOA (bromuro trimethyloctylammonium) </td><td> 30,66 mg / ml </td></tr><tr><td> TMDA (bromuro trimethyldecylammonium) </td><td> 34,07 mg / ml </td></tr><tr><td> TMDDA (bromuro trimethyldodecylammonium) </td><td> 37,48 mg / ml </td></tr><tr><td> TMTDA (bromuro trimethyltetradecylammonium) </td><td&gt; 40,89 mg / ml </td></tr><tr><td> TMHDA (bromuro trimetilesadecilammonio) </td><td> 44,31 mg / ml </td></tr><tr><td> TMODA (bromuro trimetilesadecilammonio) </td><td> 47,71 mg / ml </td></tr></tbody></table> Tabella 1. Importi di tetra-alchil sali di ammonio da utilizzare per Nafion polimero modifica. <table border="1"><tbody><tr><td> Tipo di Nafion </td><td> L&#39;attività enzimatica (U / g) </td></tr><tr><td> Buffer (no polimero) </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> Tabella 2 NAD-dipendente attività glucosio deidrogenasi immobilizzato in Nafion selezionati polimeri modificati (nota: attività immobilizzata è una funzione iniziale di attività specifica dell&#39;enzima).. <table border="1"><tbody><tr><td> Tipo di Nafion </td><td> L&#39;attività enzimatica (mU / g) </td></tr><tr><td> Buffer (no polimero) </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> Tabella 3 PQQ dipendente dalla attività glucosio deidrogenasi immobilizzato in Nafion selezionati polimeri modificati (nota: attività immobilizzata è una funzione iniziale di attività specifica dell&#39;enzima).. <table border="1"><tbody><tr><td> Tipo di Nafion </td><td> L&#39;attività enzimatica (U / g) </td></tr><tr><td> Buffer (no polimero) </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> . Rappresentativa Tabella 4 attività specifica glucosio ossidasi immobilizzata in Nafion selezionati polimeri modificati (nota: attività immobilizzata è una funzione iniziale di attività specifica dell&#39;enzima). <img alt="Figura 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> Figura 1. Schema di incorporazione TBAB Nafion in polimero e il successivo utilizzo in immobilizzazione degli enzimi. <img alt="Figura 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> Figura 2. Fotografia ottica di un primo atto co-cast film di Nafion e TBAB. Lenta asciugatura produce un pellicola trasparente, marrone chiaro copre la bottom del vassoio di pesatura.

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Nel procedimento descritto, tetra-alchil ammonio vengono utilizzati per modificare Nafion commerciale per creare micellari polimeri che possono essere utilizzati per immobilizzare e stabilizzare gli enzimi. I saggi descritti nella procedura mostrano che il polimero può essere utilizzato per immobilizzare un&#39;ampia varietà di enzimi con un&#39;elevata ritenzione di attività. Se l&#39;enzima di interesse ha una attività molto bassa o è impuro, una più alta concentrazione può essere richiesta e non devono inficia...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Nafion </td><td> Sigma-Aldrich </td><td> 70160 </td></tr><tr><td> Tetra alchilammonio bromuro di sali </td><td> Sigma-Aldrich </td><td> n / a </td></tr><tr><td> Alcool deidrogenasi </td><td> Sigma-Aldrich </td><td> A3263 </td></tr><tr><td> Nicotinammide adenin dinucleotide (NAD) </td><td> SIGMA-Aldrich </td><td> N7004 </td></tr><tr><td> Pirofosfato di sodio </td><td> Sigma-Aldrich </td><td> P8010 </td></tr><tr><td> Fenazina metosolfato (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> Glucosio ossidasi </td><td> Sigma-Aldrich </td><td> G7141 </td></tr><tr><td> 4-idrossibenzoico </td><td> Sigma-Aldrich </td><td> 240141 </td></tr><tr><td> Sodio azide </td><td> Sigma-Aldrich </td><td> S8032 </td></tr><tr><td> Perossidasi </td><td> Sigma-Aldrich </td><td> P8375 </td></tr><tr><td> 4-aminoantipirina </td><td> Sigma-Aldrich </td><td> 06800 </td></tr><tr><td> UV / Vis Spettrofotometro </td><td> Thermo </td><td> Evolution 260 Bio o Spectronic Genesys 20 </td></tr><tr><td> Vortex Genie </td><td></td><td></td></tr><tr><td> Bilancia analitica </td><td></td><td></td></tr></tbody></taBLE&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Negli ultimi dieci anni, c&#39;è stata una ricchezza di domanda di enzimi immobilizzati e stabilizzati tra biocatalisi, biosensori, e le cellule di biocarburanti. 1-3 In maggior parte delle applicazioni bioelectrochemical, enzimi o organuli sono immobilizzati su una superficie dell&#39;elettrodo con l&#39;utilizzo di un certo tipo di matrice polimerica. Questa impalcatura polimero dovrebbe tenere gli enzimi stabile e consentire la facile diffusione delle molecole e ioni in e fuori dalla matrice. La maggior parte dei polimeri utilizzati per questo tipo di immobilizzazione sono basati su poliammine o polialcoli - polimeri che imitano l&#39;ambiente naturale degli enzimi che incapsulano e stabilizzare l&#39;enzima mediante idrogeno o legame ionico. Un altro metodo per stabilizzare gli enzimi comporta l&#39;uso di micelle, che contengono regioni idrofobiche che può incapsulare e stabilizzare gli enzimi. 4,5 In particolare, il gruppo Minteer ha sviluppato un polimero basato su micellare Nafion commercialmente disponibili. 6,7 Nafionsi è un polimero che permette micellare del canale assistita diffusione di protoni e altri cationi piccoli, ma le micelle e canali sono estremamente piccoli e il polimero è molto acido solfonico dovuto catene laterali acide, il che è sfavorevole per l&#39;immobilizzazione degli enzimi. Tuttavia, quando Nafion viene miscelato con un eccesso di idrofobi sali di alchilammonio quali tetrabutilammonio bromuro (TBAB), i cationi di ammonio quaternario sostituire i protoni e gli ioni diventano contatore ai gruppi solfonato sulle catene laterali polimeriche (Figura 1). Questo crea delle micelle e canali all&#39;interno del polimero che consentono la diffusione di grandi substrati e ioni che sono necessari per la funzione enzimatica come nicotinammide adenin dinucleotide (NAD). Questo polimero Nafion modificato è stato usato per immobilizzare vari tipi di enzimi e mitocondri per l&#39;uso in biosensori e cellule biocarburanti. 8-12 Questo documento descrive una nuova procedura per effettuare questo microfonoellar membrana polimerica immobilizzazione degli enzimi in grado di stabilizzare gli enzimi. La sintesi della membrana micellare immobilizzazione dell&#39;enzima, la procedura per immobilizzare gli enzimi all&#39;interno della membrana, e saggi per studiare le attività enzimatica specifica dell&#39;enzima immobilizzato sono riportate di seguito.

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

Questo articolo descrive la procedura di sintesi di un enzima modificato in modo idrofobo membrana Nafion immobilizzazione e come immobilizzare proteine ​​e / o enzimi all&#39;interno della membrana e verificare la loro attività specifica.

Idrofobo Salt-modificato Nafion per l&#39;immobilizzazione degli enzimi e la stabilizzazione

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 Views.  University of Utah. Questo articolo descrive la procedura di sintesi di un enzima modificato in modo idrofobo membrana Nafion immobilizzazione e come immobilizzare proteine ​​e / o enzimi all'interno della membrana e verificare la loro attività specifica.

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).

Immobilizzazione batterica per Imaging di microscopia a forza atomica

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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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 piattaforma automatizzata per la scoperta degli enzimi biomassa e ottimizzazione 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.

Layer-by-layer collagene Deposizione nel Microfluidic Dispositivi per microtessuto Stabilizzazione

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 Chimica e Peptide immobilizzazione su politetrafluoroetilene per Miglioramento di adesione cellulare

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 plasmatica di consegna di un idrofobo Cargo incapsulato in un cristallo nanoparticelle Carrier Liquid

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.

Site-Directed immobilizzazione della proteina morfogenetica ossea 2 a superfici solide di 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.

Produzione di Nafion rivestite, ridotto grafene ossido/Polyaniline Chemiresistive sensore al pH Monitor in Real-Time durante la fermentazione microbica

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-immobilizzazione funzionale dei geni usando Multistep Strand Displacement Litografia

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 nanoprecipitazione per l'incapsulamento di composti idrofobi e idrofili in nanoparticelle polimeriche

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.

Immobilizzazione degli individui Live Caenorhabditis elegans utilizzando un Chip microfluidico Polydimethylsiloxane di ultra-sottile con ritenzione idrica

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