著者らは、米海軍研究、米国大豆ボード、および資金調達のために国立科学財団の事務所を認める。

1。第四級アンモニウム塩とナフィオンの変更<ol><li>約のために精力的に5パーセントw / vのナフィオン懸濁液のボトルを振ります。ナフィオン溶液中で均一にサスペンドされていることを保証するために30秒。 </li><li>ガラスバイアル（バイアル量は2.5 mLから10 mLに含まれている可能性があり）になりました再懸濁したナフィオン2 mLのピペットを。 </li><li>アルキルアンモニウム臭化物塩の3倍モル過剰（ナフィオンポリマーにスルホン酸基からの相対パス）（適切な質量を表1に示す）を測定し、ナフィオンの2 mLを含むバイアルにこれを追加します。 </li><li>渦10〜15分間1500rpmでバイアル。 </li><li>約を測定するプラスチック製秤量トレイに粘性溶液を注ぐ。 3インチ×3、計量トレイにバイアルからの残液を転送するためにピペットを使用しています。 </li><li>溶剤計量トレイの底部（Fiの黄色/茶色、透明フィルムを残して、重船外に蒸発することができますグレ2）。溶媒の蒸発速度は、それが蒸発するすべての溶媒に6時間以上がかかるようにすべきである。溶媒が蒸発が速すぎる場合は、白、無愛想な材料は、ポリマーのミセル構造が破壊されたことを示す透明フィルムの代わりに形成され、このプロシージャを再起動する必要があります。溶媒の蒸発が遅すぎる場合は、蒸発の速度が遅すぎる典型的に粘着性の、オレンジ色のゲルにつながるとは、プロシージャを再起動する必要がありますので、デ加湿器は、必要があるかもしれません。 37℃ - 溶媒蒸発のための典型的な温度範囲は20です。乾燥の実際の条件は、部屋の相対湿度と温度の関数であるが、それはゲル形成を可能にするためにミセル構造を維持するために、低速ではなく、人間が遅すぎて乾燥することが重要です。 </li><li> 18MΩcmで脱イオン水（水の10〜20 mL）で船の重量を量る記入し、カバーし、過剰なアルキルアンモニウム臭化物塩およびHBrを削除するには、12〜24時間浸漬することができます。 </li><lI&gt;水アウト（またはピペット）注ぎ、トレイたびを満たすのに十分なDI水で3回すすいでください。このステップの間にポリマーフィルムのいずれかを紛失しないように注意してください。 </li><li>ポリマーは、この時点で完全に乾燥するまで明らかに座って秤量トレイを許可する、ポリマーは、はっきりとやや脆いプラスチックフィルムでなければなりません。空気が非常に湿度が高い場合は、再度、デ加湿器は、適時に蒸発を完了する必要があるかもしれません。 </li><li>へらを使用して、慎重に計量トレイから乾燥したフィルムを除去してクリーンなガラスバイアルにそれを転送します。 </li><li>エタノール2 mLの3セラミックの混合ビーズ、4時間またはポリマーフィルムが完全に再懸濁されるまでボルテックスする。 </li></ol> 2。活性アッセイのためにTBAB-Modifiedのナフィオンに酵素の固定化<ol><li>乾燥した酵素を、1.5 mlのマイクロ遠心チューブに酵素1-10ミリグラムを量る、1〜10 mg / mLの酵素液を作成するには、100mMのpH7のリン酸緩衝液1 mLを加える。フォーrは溶液中にある酵素は、タンパク質の量を決定するためにビシンコニン酸（BCA）アッセイ13を使用して、1から10 mg / mlにタンパク質濃度をもたらすために、100mMのリン酸緩衝液の適切な量を追加します。 1から10 mg / mLのは、通常、1から50ナノモル/ mLに相当します。 </li><li> 1 mg / mLの酵素溶液120μLに、アルキルアンモニウム修飾ナフィオン溶液と、10秒間ボルテックスを60μLを追加します。 （この混合物を複製し、多数のためにスケールアップすることができます。2:1に酵素·ツー·ポリマー溶液の比率を保持します。） </li><li>ピペット60 3別の1cm 2のキュベットの底に酵素/ポリマー溶液のμL、一晩乾燥させます。 </li></ol> 3。固定化NAD-依存性脱水素酵素のアッセイ<ol><li>キュベットに、50mMのピロリン酸ナトリウム液（pH 8.8）1.3 mLを、15mMのNAD（新たに調製）、0.1 mLの水1.5 mLを加える。 </li><li> UV / VIS分光光度計（すなわちThermoScientific Evolutにキュベットを配置イオン260バイオ·サーモジェネシスSpectronic 20）340 nmの波長に設定されています。 </li><li>分光計をゼロにし、0.1mLのエタノールを加える。穏やかに5回ダウンソリューションをピペッティングにより試薬を混ぜる。空白の場合は、追加の水0.1 mLの代わりにエタノール0.1 mLを使用しています。 </li><li>試薬をキュベットに添加し、20分後にされた後5分で340 nmの吸光度を記録します。アクティビティの計算に使用することができるスロープを得るために2つのデータポイントをプロットします。 </li></ol> 4。固定PQQ依存性脱水素酵素のアッセイ<ol><li>キュベットに、リン酸ナトリウム1.5mLの液（pH7.3）、600μMPMSの200μLを追加します。 </li><li> 600nmの波長に設定UV / VIS分光光度計で、キュベットを配置し、分光計をゼロに。 </li><li> 700μLDCIPの100μLと関心のある基板（エタノール、アセトアルデヒド、グリセロール、グルコース、またはグリセルアルデヒド）の200μLを追加し、優しくsolutをピペッティングにより試薬を混ぜてイオン上下に5回。空白の場合は、200μLの水の代わりに、興味のある基板を使用しています。 </li><li>試薬をキュベットに添加し、20分後にした5分後に600 nmの吸光度を記録します。 </li></ol> 5。固定化グルコースオキシダーゼのアッセイ<ol><li>キュベットに、0.2 M のp-ヒドロキシ安息香酸、0.02％（w / v）のアジ化ナトリウム、128 Uペルオキシダーゼ、0.3mMの4 -アミノアンチピリン、1 Mリン酸カリウム、50 mMグルコースを含む溶液の2.0 mLを加える。 5回上下にピペッティングして溶液を混合。 </li><li> 510 nmの波長に設定UV / VIS分光光度計ではキュベットを配置します。 </li><li>試薬は20分後に再びキュベットに添加した後5分で、510nmで吸光度を記録します。 </li></ol> 6。代表的な結果変更されたナフィオンポリマーのミセル構造は、オリジナルの塩/ポリマーの共キャストフィルムもFasを乾燥させることにより破壊することができるトン。 図2は、正しく、透明淡褐色膜に生じる乾燥させた塩/ポリマー混合物を示しています。乾燥が速すぎて乾燥プロセスはミセル構造を破壊することができるという事実のためにポリマーの不透明な、白色のフレークにつながることができるフィルム。 変更されたナフィオンポリマーと酵素を混合し、キュベットの底面に共存キャストされた後、酵素活性アッセイは、ポリマー膜内の酵素の安定性を評価するために使用することができます。 テーブルは、2つのデヒドロゲナーゼ酵素の2-4はアッセイの結果とグルコースオキシダーゼは、それぞれ、さまざまな変更されたナフィオン膜に固定化した。変更されたナフィオンポリマーが実際に特定の酵素（superactivityと呼ばれる）の活性を高めることができることを示す、緩衝液中の酵素対固定されている酵素の活性が高いことに注意してください。他の酵素は、ポリマーにそれらを固定するときに、その特定の活性を低下させる輸送の制限があります（その基板）非常に大きい巨大分子である、すなわちセルラーゼおよびアミラーゼ、。 <table border="1"><tbody><tr><td> 使用される第四級アンモニウム塩 </td><td> 3倍過剰 </td></tr><tr><td> T3A（テトラブロミド） </td><td> 32.37 mg / mlの</td></tr><tr><td> TBAB（テトラブチルアンモニウムブロマイド） </td><td> 39.19 mg / mlの</td></tr><tr><td> TPAB（テトラペンチブロマイド） </td><td> 46.​​01 mg / mlの</td></tr><tr><td> TEHA（triethylhexylammoniumブロマイド） </td><td> 32.37 mg / mlの</td></tr><tr><td> TMHA（trimethylhexylammoniumブロマイド） </td><td> 27.25 mg / mlの</td></tr><tr><td> TMOA（trimethyloctylammoniumブロマイド） </td><td> 30.66 mg / mlの</td></tr><tr><td> TMDA（trimethyldecylammoniumブロマイド） </td><td> 34.07 mg / mlの</td></tr><tr><td> TMDDA（trimethyldodecylammoniumブロマイド） </td><td> 37.48 mg / mlの</td></tr><tr><td> TMTDA（trimethyltetradecylammoniumブロマイド） </td><tD&gt; 40.89 mg / mlの</td></tr><tr><td> TMHDA（trimethylhexadecylammoniumブロマイド） </td><td> 44.31 mg / mlの</td></tr><tr><td> TMODA（trimethyloctadecylammoniumブロマイド） </td><td> 47.71 mg / mlの</td></tr></tbody></table> 表1。ナフィオンポリマー改質に使用するテトラアルキルアンモニウム塩の量。 <table border="1"><tbody><tr><td> ナフィオンのタイプ </td><td> 酵素活性（U / g）を </td></tr><tr><td>バッファ（NOポリマー） </td><td> 16.63±8.11 </td></tr><tr><td>ナフィオン（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> 表2選択され変更されたナフィオンポリマー（注：固定化活性は酵素の初期比活性の関数である）に固定化したNAD依存性グルコース脱水素酵素活性。 <table border="1"><tbody><tr><td> ナフィオンのタイプ </td><td> 酵素活性（MU / g）を </td></tr><tr><td>バッファ（NOポリマー） </td><td> 7.18±0.51 </td></tr><tr><td>ナフィオン（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> 表3 PQQ依存性グルコース脱水素酵素活性は、（注：固定化活性は酵素の初期比活性の関数である）を選択して変更されたナフィオンポリマーに固定化した。 <table border="1"><tbody><tr><td> ナフィオンのタイプ </td><td> 酵素活性（U / g）を </td></tr><tr><td>バッファ（NOポリマー） </td><td> 103.61±3.15 </td></tr><tr><td>ナフィオン（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> 表4代表的なグルコースオキシダーゼ比活性は、選択され変更されたナフィオンポリマー（：固定化活性が酵素の初期比活性の関数であり、注意してください）に固定することができる。 <img alt="図1" src="/files/ftp_upload/3949/3949fig1.jpg" /> 図1ナフィオンポリマーと酵素の固定化のその後の使用にTBABの取り込みの模式図。 <img alt="図2" src="/files/ftp_upload/3949/3949fig2.jpg" /> 図2ナフィオンとTBABの最初の共同キャストフィルムの光学写真。遅乾収量boを覆う透明な、ライトブラウンフィルム秤量トレイのttom。

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利害の衝突が宣言されません。

記載されている手順では、テトラアルキルアンモニウム塩は酵素を固定化と安定化するために使用することができミセルポリマーを作成するために商業的なナフィオンを変更するために使用されています。アッセイは、ポリマーは活性の高い保持と酵素の多種多様を固定するために使用することができる手順番組で説明しています。興味のある酵素が非常に低い活性を有する、または不純で?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ナフィオン</td><td> Sigma-Aldrich社</td><td> 70160 </td></tr><tr><td>テトラアルキルアンモニウム臭化物塩</td><td> Sigma-Aldrich社</td><td> N / A </td></tr><tr><td>アルコール脱水素酵素</td><td> Sigma-Aldrich社</td><td> A3263 </td></tr><tr><td>ニコチンアミドアデニンジヌクレオチド（NAD） </td><td> Simga-Aldrich社</td><td> N7004 </td></tr><tr><td>ピロリン酸ナトリウム</td><td> Sigma-Aldrich社</td><td> P8010 </td></tr><tr><td>フェナジンメトサルフェート（PMS） </td><td> Sigma-Aldrich社</td><td> P9625 </td></tr><tr><td> （DCIP）2,6 - ジクロロ</td><td> SIGMA-Aldrich社</td><td> D1878 </td></tr><tr><td>ブドウ糖酸化酵素</td><td> Sigma-Aldrich社</td><td> G7141 </td></tr><tr><td> 4 - ヒドロキシ安息香酸</td><td> Sigma-Aldrich社</td><td> 240141 </td></tr><tr><td>アジ化ナトリウム</td><td> Sigma-Aldrich社</td><td> S8032 </td></tr><tr><td>ペルオキシダーゼ</td><td> Sigma-Aldrich社</td><td> P8375 </td></tr><tr><td> 4 - アミノアンチピリン</td><td> Sigma-Aldrich社</td><td> 06800 </td></tr><tr><td> UV / VIS分光光度計</td><td>サーモ</td><td>進化260バイオまたはSpectronicジェネシス20 </td></tr><tr><td>渦魔神</td><td></td><td></td></tr><tr><td>化学てんびん</td><td></td><td></td></tr></tbody></taBLE&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

過去10年間、生体触媒、バイオセンサー、バイオ燃料細胞を含む固定し、安定した酵素を、アプリケーションの豊富がありました。最もbioelectrochemicalアプリケーションでは1-3、酵素や細胞小器官は、いくつかのタイプのを利用して電極表面に固定化されているポリマーマトリックス。このポリマー足場は、酵素の安定性を維持し、マトリックスの内と外の分子やイオンの容易な拡散を可能にする必要があります。彼らは水素原子またはイオン結合を介して酵素をカプセル化し、安定させることが酵素の自然環境を模倣ポリマー - 固定化のこのタイプに使用されるほとんどのポリマーは、ポリアミンまたはポリアルコールに基づいています。別の安定化酵素の方法酵素をカプセル化し、安定させることができる疎水性領域を含むミセルの使用を含む。特に、4,5、Minteerグループは、市販のナフィオンに基づくミセルポリマーを開発しました。6,7ナフィオン自体は、陽子や他の小さな陽イオンチャネルアシスト拡散することができミセル重合体であるが、ミセルとのチャネルは非常に小さいとポリマーが酵素固定化のために不利であるスルホン酸側鎖のために非常に酸性である。しかしながら、ナフィオンはそのような臭化テトラブチルアンモニウム（TBAB）のような疎水性のアルキルアンモニウム塩の過剰と混合されたときに、第四級アンモニウムカチオンは、プロトンを交換し、ポリマーの側鎖（ 図1）スルホン酸基のカウンターイオンとなる。このようなニコチンアミドアデニンジヌクレオチド（NAD）などの酵素機能するために必要な大型基板とイオンの拡散を可能にするポリマー内でより大きなミセルとチャネルでこの結果。この変更されたナフィオンポリマーは、バイオセンサーやバイオ燃料のセルで使用するために、さまざまな酵素の種類と同様にミトコンドリアを固定するために使用されています。8月12日本論文では、このマイクを行うための新たな手順を説明します酵素を安定させることができるellarポリマーの酵素固定化膜。ミセルの酵素固定化膜の合成、膜内に酵素を固定化するための手順、及び固定化酵素の酵素比活性を研究するためのアッセイは、以下に詳述されています。

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

この資料では、疎水性修飾ナフィオン酵素固定化膜方法と膜内のタンパク質および/または酵素を固定化とその特異的活性をテストするを合成するための手順を説明します。

酵素の固定化と安定化のための疎水性の塩で修飾ナフィオン

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 ビュー.  University of Utah. この資料では、疎水性修飾ナフィオン酵素固定化膜方法と膜内のタンパク質および/または酵素を固定化とその特異的活性をテストするを合成するための手順を説明します。

ビデオ: 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).

原子間力顕微鏡によるイメージングのための細菌の固定化

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 ...

生物工学

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：バイオマスの酵素の発見とカクテルの最適化のための自動化プラットフォーム

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安定化のためのマイクロ流体デバイスの層ごとのコラーゲン沈着

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.

改善された細胞接着のためのポリテトラフルオロエチレンにウェットケミストリーおよびペプチドの固定化

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.

液晶ナノ粒子キャリアにおける疎水性貨物カプセル化の標的細胞膜配達

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.

骨形成タンパク質 2 固体表面にクリックケミストリーによるサイト向け固定

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.

ナフィオン被覆の製造、削減グラフェン酸化物/ポリアニリン Chemiresistive 微生物発酵中にリアルタイムでモニター ph センサー

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.

多段鎖変位リソグラフィーを用いた遺伝子の機能的な面を固定化

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.

高分子ナノ粒子の疎水性および親水性化合物のカプセル化のため NanoPrecipitation をフラッシュします。

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.

極薄ポリジメチルシロキサン マイクロ流体チップを用いた保水ライブ線虫個人の固定化

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