Yazarlar Donanma Araştırma, Birleşik Soya Kurulu ve finansmanı için Ulusal Bilim Vakfı Office kabul ederler.

1. Kuaterner Amonyum Tuzları ile Nafion Modifikasyonu <ol><li> Yaklaşık kuvvetlice% 5 w / v Nafion süspansiyon bir şişe çalkalanır. Bu Nafion sağlamak için 30 saniye çözelti içinde eşit şekilde süspanse edilir. </li><li> Bir cam şişeye (şişeye hacmi 2.5 ml ila 10 ml içerebilir) içine artık tekrar askıya Nafion 2 mL dışarı Pipeti. </li><li> Bir 3-kat molar fazlalıktan (Nafion polimer üzerindeki sülfonik asit grupları oranla) alkil amonyum bromür tuzu (uygun kütleler Tablo 1 &#39;de gösterilmiştir) ile ölçülür ve Nafion 2 mL ihtiva eden bir şişeye, bu ekleyin. </li><li> 10-15 dakika 1500 rpm&#39;de Vortex flakon. </li><li> Yaklaşık ölçen bir plastik tartım tepsisine viskoz solüsyonu dökün. 3 x 3 inç ve tartım tepsiye tüpten kalan çözüm aktarmak için bir pipet kullanın. </li><li> Çözücüler tartım tepsinin alt (Fi sarı / kahverengi, şeffaf film bırakarak, tartmak tekne dışarı buharlaşmasına izinGure 2). Çözücü buharlaştırma ile oranı da buharlaştırmak için tüm solvent için birden fazla 6 saat sürer ki bu olmalıdır. Çözücü buharlaşır çok hızlı, bir beyaz, kabuklu malzemenin polimerin micellar yapısı tahrip olmuştur belirten bir saydam film yerine oluşturacak ve prosedür tekrar başlaması gerekir. Solvent buharlaşması çok yavaş ise buharlaşma çok yavaş tipik bir yapışkan, portakal jel yol ve yordamı yeniden başlamalıdır, çünkü bir de nemlendirici, gerekli olabilir. 37 ° C - solvent buharlaşması için tipik sıcaklık aralıkları 20 olan Kurutma için gerçek koşulları odasında bağıl nem ve sıcaklık bir fonksiyonu vardır, ama önemli olan jel oluşumuna imkan vermek için kuruma misel yapısını korumak için yavaş olmak, ama çok yavaş olduğunu. </li><li> Dolgu 18M Ωcm de-iyonize su (su, 10-20 ml), kapak ile tekne tartmak ve fazla alkil amonyum bromür tuzları ve HBr kaldırmak için 12-24 saat suda bekletiniz. </li><li&gt; suyu (ya da pipet) dökün ve tepsiyi her zaman doldurmak için yeterli DI su ile 3 kez yıkayın. Bu adım esnasında polimer filmin herhangi bir kaybetmemek için dikkatli olun. </li><li> Polimer Bu noktada tamamen kuruduktan sonra ortaya oturmak tartım tepsisi İzin, polimer açık ve biraz kırılgan plastik film olmalıdır. Hava çok rutubetli Yine, bir nem-zamanında buharlaşma tamamlamak için gerekli olabilir. </li><li> Spatula kullanarak, dikkatli bir şekilde tartım tepsiden kurutulmuş filmi çıkarın ve temiz bir cam şişeye aktarın. </li><li> 2 mL etanol ve 3 seramik karıştırma boncukları ve 4 saat boyunca vorteks eklemek veya polimer filmi tamamen yeniden süspanse kadar. </li></ol> 2. Aktivite Tahliller için TBAB-Modifiye Nafion içine Enzimlerin immobilizasyonu <ol><li> Kuru bir enzim, bir 1.5 ml mikrosantrifüj tüp içine enzim 1-10 mg tarttın, ve bir 1-10 mg / ml enzim çözeltisi oluşturmak için 100 mM pH 7 fosfat tampon 1 ml ilave edilir. For çözelti içinde bir enzim, protein miktarını belirlemek için, bir bicinchoninic asit (BCA) test 13 kullanmak ve 1-10 mg / mL kadar protein konsantrasyonu getirmek için 100 mM fosfat tampon maddesi ile uygun bir miktarda ilave edin. 1-10 mg / mL, genellikle 1-50 nanomol / mL &#39;ye karşılık gelir. </li><li> 1 mg / ml enzim çözeltisinin 120 uL için, 10 saniye için 60 alkil amonyum ile modifiye Nafion ® çözeltisi ile uL, ve girdap ekleyebilir. (Bu karışım çoğaltır büyük sayılar için kadar ölçeklendirilebilir. 2:1 enzim-to-polimer çözeltisi oranı tutun.) </li><li> Pipet 60 3 ayrı 1 cm 2 cuvettes dibine enzim / polimer çözüm uL, ve geceleme kurumasını bekleyin. </li></ol> 3. İmmobilize NAD-bağımlı Dehidrogenaz Enzim Testi <ol><li> Küvetine, 50 mM sodyum pirofosfat (pH 8.8) ve 1.3 mL, 15 mM NAD (taze hazırlanmış) ve 1,5 mL ve 0,1 mL su ilave edilir. </li><li> UV / Vis spektrofotometre (yani ThermoScientific Evolut de küvet koyuniyon 260 Bio ve Thermo Spectronic Genesys 20) 340 nm dalga boyunda ayarlanmış. </li><li> Spektrometre sıfır, ve daha sonra 0.1 ml etanol ilave edin. Hafifçe 5 kez aşağı çözümü pipetleme tarafından reaktifleri karıştırın. Boş için ek su 0,1 ml yerine etanol 0,1 ml kullanın. </li><li> Reaktifler sonra küvet ve 20 dakika sonra ilave edildi 5 dakikada 340 nm&#39;de absorbans kaydediniz. Aktivite hesaplamalar için kullanılabilecek bir eğim elde etmek için iki veri noktaları çizilir. </li></ol> 4. İmmobilize PQQ-Bağımlı dehidrogenazlar miktar tayini <ol><li> Küvet için, sodyum fosfat 1.5 mL (pH 7.3) ve 600 mM PMS 200 ul ekleyin. </li><li> 600 nm dalga boyunda ayarlanmış bir UV / Vis spektrofotometre içinde küvet yerleştirin ve sonra spektrometre sıfıra. </li><li> 700 uL DCIP 100 uL ve faiz substrat (etanol, asetaldehit, gliserol, glikoz veya gliseraldehit) 200 uL ekleyin ve hafifçe solut pipetleme tarafından reaktifler karıştırıniyon aşağı yukarı 5 kez. Boş için 200 su uL yerine ilgi substrat kullanın. </li><li> Reaktifler sonra küvet ve 20 dakika sonra ilave edildi 5 dakikada 600 nm&#39;de absorbans kaydediniz. </li></ol> 5.. İmmobilize Glukoz Oksidaz miktar tayini <ol><li> Küvetine, 0.2 M, p-hidroksibenzoik asit,% 0.02 (w / v) sodyum azid, 128 U peroksidaz, 4-aminoantipirin 0.3 mM, 1 M potasyum fosfat, ve 50 mM glukoz ihtiva eden bir çözeltiden 2.0 ml ilave edilir. 5 kez aşağı yukarı pipetleme tarafından çözüm karıştırın. </li><li> 510 nm dalga boyunda bir UV / Vis spektrofotometre sette küvet yerleştirin. </li><li> Reaktifler sonra 20 dakika küvet içinde tekrar ilave edilmiştir sonra 5 dakika 510 nm&#39;de absorbans kaydediniz. </li></ol> 6. Temsilcisi Sonuçlar Modifiye Nafion polimerin micellar yapısı orijinal tuzu / polimer co-döküm filmin çok Haldun&#39;un kurutularak bozulabilmektedirt. Şekil 2 doğru şeffaf, açık kahverengi bir tabakaya yolaçan kurutulmuş olan bir tuzu / polimer karışımı gösterir. Kurur çok hızlı kurutma işlemi misel yapısı tahrip etme nedeni polimer opak, beyaz pul neden olabilir bir film. Modifiye Nafion polimer ve enzim karıştırılır ve bir küvet alt üzerinde eş-atılmıştır sonra, enzimatik testler aktivitesi polimer filmi içinde enzim stabilite değerlendirmek için kullanılabilir. Tablolar iki dehidrogenaz enzimlerin 2-4 kez deneyi sonuçları ve glukoz oksidaz sırasıyla çeşitli modifiye Nafion filmlerinde immobilize. Modifiye Nafion polimerler, aslında, belirli enzimler (superactivity olarak adlandırılır) aktivitesi artırabilir gösteren, tampon çözeltisi içinde enzimlerin vs immobilize edilen enzimler daha yüksek aktivite not edin. Diğer enzimler polimer onları hareketsiz zaman kendi özel aktivite azaltmak ulaşım sınırlamaları vardır (olan yüzeyler) oldukça büyük makromoleküller yani selülazlar ve amilazlar. <table border="1"><tbody><tr><td> Kullanılan kuaterner amonyum tuzu </td><td> 3 kat daha fazla </td></tr><tr><td> T3a (tetrapropylammonium bromür) </td><td> 32,37 mg / ml </td></tr><tr><td> TBAB (tetrabutilamonyum bromür) </td><td> 39,19 mg / ml </td></tr><tr><td> TPAB (tetrapentylammonium bromür) </td><td> 46,01 mg / ml </td></tr><tr><td> TEHA (triethylhexylammonium bromür) </td><td> 32,37 mg / ml </td></tr><tr><td> TMHA (trimethylhexylammonium bromür) </td><td> 27.25 mg / ml </td></tr><tr><td> TMOA (trimethyloctylammonium bromür) </td><td> 30,66 mg / ml </td></tr><tr><td> TMDA (trimethyldecylammonium bromür) </td><td> 34.07 mg / ml </td></tr><tr><td> TMDDA (trimethyldodecylammonium bromür) </td><td> 37.48 mg / ml </td></tr><tr><td> TMTDA (trimethyltetradecylammonium bromür) </td><td&gt; 40,89 mg / ml </td></tr><tr><td> TMHDA (trimethylhexadecylammonium bromür) </td><td> 44,31 mg / ml </td></tr><tr><td> TMODA (trimethyloctadecylammonium bromür) </td><td> 47,71 mg / ml </td></tr></tbody></table> Tablo 1. Tetra-alkil amonyum tuzları edilmiştir Nafion polimer modifikasyonu için kullanımı. <table border="1"><tbody><tr><td> Nafion Tipi </td><td> Enzim aktivitesi (U / g) </td></tr><tr><td> Tampon (hayır polimer) </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> Tablo 2 seçilen modifiye Nafion polimerler (not: immobilize aktivitesi enzim başlangıç ​​spesifik aktivite bir fonksiyonudur) immobilize NAD-bağımlı glukoz dehidrogenaz aktivitesi.. <table border="1"><tbody><tr><td> Nafion Tipi </td><td> Enzim aktivitesi (mU / g) </td></tr><tr><td> Tampon (hayır polimer) </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> Tablo 3 PQQ-glukoz dehidrogenaz bağımlı aktivitesi seçilen modifiye Nafion polimerler (not: immobilize aktivitesi enzim başlangıç ​​spesifik aktivite bir fonksiyonudur) immobilize.. <table border="1"><tbody><tr><td> Nafion Tipi </td><td> Enzim aktivitesi (U / g) </td></tr><tr><td> Tampon (hayır polimer) </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> . Tablo 4 Temsilcisi glukoz oksidaz spesifik aktivite seçilen modifiye Nafion polimerler (: immobilize enzim aktivitesi ilk spesifik aktivitelerinin bir fonksiyonudur not) immobilize. <img alt="Şekil 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> Şekil 1. Nafion polimer ve enzim immobilizasyonu daha sonraki kullanıma TBAB şirketleşme şematik. <img alt="Şekil 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> Şekil 2. Nafion ve TBAB ilk co-döküm filmlerin optik fotoğraf. Verimleri bo kapsayan şeffaf, açık kahverengi film kurutma Yavaştartım tepsi ttom.

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Çıkar çatışması ilan etti.

Açıklanan prosedür içinde, tetra-alkil amonyum tuzları enzimler hareketsiz ve stabilize etmek için kullanılabilir micellar polimerler oluşturmak için ticari Nafion değiştirmek için kullanılır. Testler polimer aktivitesi yüksek bir tutma ile enzimler çeşitli immobilize etmek için kullanılan bir prosedürü göstermektedir tarif. Söz konusu enzimi çok düşük bir aktiviteye sahiptir veya saf olmayan ise, daha yüksek bir konsantrasyonu gerekli olabilir ve 10 mg / ml &#39;den daha büyük konsantrasyo...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Nafion </td><td> Sigma-Aldrich </td><td> 70160 </td></tr><tr><td> Tetra alkilamonyum bromit tuzlarıdır </td><td> Sigma-Aldrich </td><td> n / a </td></tr><tr><td> Alkol dehidrogenaz </td><td> Sigma-Aldrich </td><td> A3263 </td></tr><tr><td> Nikotinamid adenin dinükleotid (NAD) </td><td> Simga-Aldrich </td><td> N7004 </td></tr><tr><td> Sodyum pirofosfat </td><td> Sigma-Aldrich </td><td> P8010 </td></tr><tr><td> Fenazin methosulfate (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> Glukoz oksidaz </td><td> Sigma-Aldrich </td><td> G7141 </td></tr><tr><td> 4-hidroksibenzoik asit </td><td> Sigma-Aldrich </td><td> 240141 </td></tr><tr><td> Sodyum azid </td><td> Sigma-Aldrich </td><td> S8032 </td></tr><tr><td> Peroksidaz </td><td> Sigma-Aldrich </td><td> P8375 </td></tr><tr><td> 4-aminoantipirin </td><td> Sigma-Aldrich </td><td> 06800 </td></tr><tr><td> UV / Vis Spektrofotometre </td><td> Thermo </td><td> Evrim 260 Bio ya Spectronic Genesys 20 </td></tr><tr><td> Vortex Genie </td><td></td><td></td></tr><tr><td> Analitik terazi </td><td></td><td></td></tr></tbody></table&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Son on yılda, biyokataliz, biyosensörler ve biyo yakıt pilleri de dahil olmak üzere tutuklanmış ve stabilize enzimleri için uygulama bir zenginlik olmuştur. En bioelectrochemical uygulamalarda 1-3, enzim veya organel bir çeşit kullanımı ile bir elektrot yüzeyine immobilize edilir polimer. Bu polimer iskele enzimler stabil tutmak ve matriks dışında molekül ve iyonları basit difüzyon için izin vermelidir. Immobilizasyon bu tür polimerler için kullanılan en poliaminler ya polialkollerdir dayanmaktadır - bunlar, hidrojen veya iyonik bağ yoluyla enzim saklanması ve stabilize bu enzimin doğal ortamda taklit eden polimerler. Enzimlerin stabilitesi için başka bir yöntem enzimler saklanması ve stabilize hidrofobik bölgeler içermesi miseller, kullanımını içerir. Özellikle 4,5, Minteer grubu, piyasada bulunan Nafion dayanan bir micellar polimer geliştirmiştir. 6,7 Nafionkendisi proton ve diğer küçük katyonlar arasında kanalı-assisted difüzyonu için izin veren bir micellar polimer, fakat miselleri ve kanallar son derece küçük olduğu ve polimer enzim harekersizliği için elverişsiz olduğu sülfonik asit yan zincirleri, nedeniyle çok asittir. Bununla birlikte, bu tür Nafion tetrabutilamonyum bromür (TBAB) gibi hidrofobik alkil amonyum tuzları bir fazlası ile karıştırıldığı zaman, kuaterner amonyum katyonları ve proton yerine polimer yan zincirleri (Şekil 1) üzerine sülfonat gruplarına karşı iyonların hale gelir. Böyle nikotinamid adenin dinükleotid (NAD) gibi enzimatik işlevi için gerekli olan geniş bir substrat ve iyon difüzyonu için izin vermek polimer içindeki büyük miselleri ve kanalları ile sonuçlanır. Bu modifiye Nafion polimer biyosensör ve biyo hücreleri kullanılmak üzere pek çok değişik enzim türleri gibi mitokondri immobilize etmek için kullanılmaktadır. 8-12 Bu kağıt Bu mikrofon yapmak için yeni bir yöntem açıklanmaktadırenzimler stabilize ellar polimer enzim immobilizasyon membran. Misel enzim immobilizasyon membran, membran içindeki enzimler immobilizing için prosedür ve immobilize enzimin enzim spesifik aktivitesi eğitimi için tahlillerin sentezi aşağıda ayrıntılı olarak açıklanmıştır.

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

Bu makale, hidrofobik olarak modifiye edilmiş Nafion enzim immobilizasyon membranı ve nasıl membran içindeki protein ve / veya enzim hareketsiz ve spesifik aktivite için test etmek için sentez prosedürü tarif edecektir.

Enzim immobilizasyonu ve stabilizasyonu için hidrofobik Tuz-modifiye Nafion

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

hydrophobic-salt-modified-nafion-for-enzyme-immobilization

Research

JoVE Journal

Bioengineering

16.1K Görüntüleme.  University of Utah. Bu makale, hidrofobik olarak modifiye edilmiş Nafion enzim immobilizasyon membranı ve nasıl membran içindeki protein ve / veya enzim hareketsiz ve spesifik aktivite için test etmek için sentez prosedürü tarif edecektir.

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).

Atomik Kuvvet Mikroskopi Görüntüleme Bakteriyel İmmobilizasyon

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 ...

Biyomühendislik

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: Biyokütle Enzim Discovery ve Kokteyl Optimizasyonu Otomatik Platform

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.

Microtissue Sabitleme için mikroakışkan Cihazlar Katman-katman Kolajen Biriktirme

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.

Geliştirilmiş Hücre yapışma Politetrafloroetilen Islak Kimya ve Peptit İmmobilizasyonu

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.

Sıvı Kristal Nanopartikül Carrier bir Hidrofobik Kargo Encapsulated, hedeflenen Plazma Zarı Teslimat

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 yönettiği immobilizasyon kemik morfogenetik protein 2 katı yüzeyler için tıklayın kimya tarafından

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.

Nafion kaplı imalatı, mikrobiyal fermantasyon sırasında gerçek zamanlı monitör pH grafen oksit/Polyaniline Chemiresistive sensör azaltılmış

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.

Genlerin Multistep Strand deplasman litografi kullanarak işlevsel yüzey immobilizasyon

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.

Hidrofobik ve hidrofilik bileşikler polimer nano tanecikleri Kapsülleme için NanoPrecipitation flash

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.

Bir Ultra-thin Polydimethylsiloxane mikrosıvısal çip su tutma ile kullanarak canlı Caenorhabditis elegans bireylerin immobilizasyon

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