この作品は、国立科学財団FIBR（NSF EF-0623664）とIDBR（NSF DBI-0754570）でサポートされています。この作品は、部分的に協力協定PHY 0120999の下でバイオフォトニクス、カリフォルニア大学デ​​ービス校で管理されるNSF科学技術センター、センターによって管理国立科学財団FIBRグラント0623664からの資金によってサポートされていました。リサLapidusは、博士の研究バローズウェルカム基金からの科学的界面におけるキャリア賞によって部分的にサポートされています。

<ol>
	<li>Eaton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+and+mechanisms+in+protein+folding.">Fast kinetics and mechanisms in protein folding.</a> Annual Review of Biophysics and Biomolecular Structure. 29, 327-359 (2000).</li><li>Hertzog, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtomole+mixer+for+microsecond+kinetic+studies+of+protein+folding.">Femtomole mixer for microsecond kinetic studies of protein folding.</a> Analytical Chemistry. 76, 7169-7178 (2004).</li><li>Hertzog, D. E., Ivorra, B., Mohammadi, B., Bakajin, O., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+microfluidic+mixer+for+studying+protein+folding+kinetics.">Optimization of a microfluidic mixer for studying protein folding kinetics.</a> Analytical Chemistry. 78, 4299-4306 (2006).</li><li>Yao, S., Bakajin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+Mixing+Time+and+Mixing+Uniformity+in+Devices+Designed+for+Studies+of+Protein+Folding+Kinetics.">Improvements in Mixing Time and Mixing Uniformity in Devices Designed for Studies of Protein Folding Kinetics.</a> Analytical Chemistry. 79, 5753-5759 (2007).</li><li>Lapidus, L. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+protein+folding+kinetics+studied+by+ultrarapid+mixing.">Submillisecond protein folding kinetics studied by ultrarapid mixing.</a> PNAS. 94, 1779-1784 (1997).</li><li>Shastry, M. C. R., Luck, S. D., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous-flow+capillary+mixing+method+to+monitor+reactions+on+the+microsecond+time+scale.">A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.</a> Biophysical Journal. 74, 2714-2721 (1998).</li><li>Pollack, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compactness+of+the+denatured+state+of+a+fast-folding+protein+measured+by+submillisecond+small-angle+x-ray+scattering.">Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 10115-10117 (1999).</li><li>Takahashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+cytochrome+c+initiated+by+submillisecond+mixing.">Folding of cytochrome c initiated by submillisecond mixing.</a> Nature Structural Molecular Biology. 4, 44-50 (1997).</li><li>Knight, J. B., Vishwanath, A., Brody, J. P., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+focusing+on+a+silicon+chip:+Mixing+nanoliters+in+microseconds.">Hydrodynamic focusing on a silicon chip: Mixing nanoliters in microseconds.</a> Physical Review Letters. 80, 3863-3866 (1998).</li><li>DeCamp, S. J., Naganathan, A. N., Waldauer, S. A., Bakajin, O., Lapidus, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Downhill+Folding+of+lambda-Repressor+in+a+Microfluidic+Mixer.">Direct Observation of Downhill Folding of lambda-Repressor in a Microfluidic Mixer.</a> Biophysical Journal. 97, 1772-1777 (2009).</li><li>Waldauer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruggedness+in+the+folding+landscape+of+protein+L.">Ruggedness in the folding landscape of protein L.</a> Hfsp Journal. 2, 388-395 (2008).</li><li>Shastry, M. C. R., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+barrier-limited+protein+folding+kinetics+on+the+microsecond+time+scale.">Evidence for barrier-limited protein folding kinetics on the microsecond time scale.</a> Nature Structural Biology. 5, 385-392 (1998).</li><li>Uzawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+and+search+dynamics+of+apomyoglobin+folding+revealed+by+submillisecond+observations+of+alpha-helical+content+and+compactness.">Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 1171-1176 (2004).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+Multiple+Folding+Pathways+for+the+Villin+Headpiece+Subdomain.">Evidence of Multiple Folding Pathways for the Villin Headpiece Subdomain.</a> The Journal of Physical Chemistry B. 115, 12632-12637 (2011).</li></ol>

我々は、開示することは何もありません。

それは長い生体分子の分子プロセスはピコ秒から秒に至る時間スケールで起こることが認識されているため、迅速な混合が長年のタンパク質の折り畳みのフィールドの開発を目標としてきました。従来のストップトフローミキサーは、主に乱流によって制限される1から5ミリ秒のデッドタイムを持っています。 30から300マイクロ秒の混合時間を持つ乱流連続フローミキサーは少数のグループで、過去15年間にわたって開発が、一般的にこれらの混合時間を達成するために、高流量を必要とするため、サンプル6-8の多くを使用しています。 このプロトコルは、層流領域で急激な希釈を達成するためのマイクロ流体混合チップの使用方法について説明します。そこにこの政権内での作業に複数の利点がありますが、プライマリは1つが混合応答を変更することができます高精度で全体の混合過程をシミュレートする機能があります。シンプルなGEOMで他のグループによって開発されたT-ミキサーetriesは、主にチャネル9から10のサイズによって制限された100から200μsの混合時間を実証した。 10μmの騎士にチャンネルのサイズをスケーリングすることにより約10μsの11に混合時間を減少させた。ヤオとBakajinは、ジオメトリの小さな変化は、混合時間4の大幅な改善につながる可能性があることを示した。しかし、その作業は、混合のその加速度も同様に遅い段階につながる可能性が示された。この作品は12月13日に使用されるデザインは変性剤濃度の遅い指数関数的減衰を最小限に抑えるために、また、DRIEエッチング機能は、より再現性のために参照4の2つの最高のデザインとの兼ね合いで決まります。 混合時間の定義を介して混合超高速の分野でいくつかの論争がありました。 &quot;混合時間&quot;と &quot;デッドタイム&quot;の違いを認識することが重要です。デッドタイム中にmeasuremen時間として乱流ミキサーで定義されていますtは作ることができないと、実際には、実際の混合時間よりも大幅に長くなる場合があります。これは通常、種々の濃度の擬似一次二分子反応（例えば、N bromosuccinateによるトリプトファン蛍光の消光など）のいくつかの測定を行うことによって決定されます。観測された指数減衰はt = 0秒と仮定し、単一の値に収束するために取り付けられており、そのポイントと最初の測定点間の時間がデッドタイムであるされています。デッドタイムの​​み、混合時間がありませんので、層流ミキサーでは、測定値は、混合プロセス中に行うことができます。混合時間は、単に十分な均一性に到達するまで、2つのソリューションを結合する時間です。我々はこれまでに10分の90時間、非混合値の10％から90％まで減少させる変性剤の濃度の時間になるように混合時間を定義しています。しかし、ミキシングカーブの形状は、小さな指数成分を有する減衰の尾を持つ完全に対称ではありません。さらに、タンパク質のフォールディングは持って変性剤濃度の非常に非線形依存性その折りには、多くの場合、変性剤のみ2倍削減されている場合でも開始できるようにします。そこで我々は、より最近では80％変性剤濃度を低減するための時間として、混合時間を定義しています。確かに他の定義は、実験の要件に応じて使用することができます。 タンパク質の折りたたみを研究するために、このミキサーの使用が一貫して驚くべき結果を明らかにした。シトクロムcやアポミオグロビンの折り畳みが長く連続した流れミキサーを持つ複数の折り畳みステップと初期の結果を持って研究されている〜100μsのタイムスケール10,14-15で発生する崩壊を示した。このミキサーで我々の測定は（遅い相に続いて、ミキサーの混合時間内に、非常に高速で、おそらく非特異的、崩壊（などのTrpの蛍光スペクトルシフトにより測定）は、このスケールの少なくとも2つのステップがあるように見せ総Trpの発光の消光によって測定される）可能性がfであることネイティブ構造5のIRST形成。また、このタンパク質は2ステートフォルダ13であるという従来の解釈を覆す、プロテインLのB1ドメインに50μsのプロセスを観察した。 この急速なミキサーの利点は、通常はナノ秒T-ジャンプの楽器で測定可能なされている最速の折りたたみ、タンパク質の折りたたみを調べることができるということです。 T-ジャンプは、通常、タンパク質の融点に近い温度緩和を折る/展開の観察を必要とするため、全人口の折りたたみに続くことはありません。このミキサーで我々はTrp総排出量とスペクトルシフトの両方でγ-リプレッサーの折りたたみ（λ6から86）を見ていると蛋白質は、以前のTにアクセスできなかった強い折り畳み条件の下で折りへの障壁を持っていないことを示す証拠を発見した·ジャンプの測定12。最後に、ビリンのかぶとHP-35、Tのいずれかの折りたたみを測定した彼最速のフォルダは、まだ測定し、混合後に測定した折りたたみ式の速度が同じ条件で16歳未満のT-ジャンプによって測定より〜5倍遅いことがわかった。これは、折り畳み速度が分野における主要な前提を覆す、開始条件に依存していることを示唆している。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> AZ P4110レジスト</td><td> AZエレクトロニックマテリアル</td><td></td><td></td></tr><tr><td> AZ 400 K開発</td><td> AZエレクトロニックマテリアル</td><td></td><td></td></tr><tr><td>ベイカーPRS 2000フォトレジストストリッパー</td><td> Avantorのパフォーマンス·マテリアル</td><td></td><td></td></tr><tr><td> AquaBond </td><td> AquaBondテクノロジーズ</td><td> AquaBond 55 </td><td></td></tr><tr><td> 500μmのカバーウエハ</td><td> SENSOR準備サービス</td><td></td><td> Ø：100ミリメートル±0.5ミリメートル 板厚：0.5 mm±0.025ミリメートル 標準公差（記述のない限り） 表面仕上げ：SI：4-8A、SII：8-12A 干潟：OneプライマリSEMI-STD; Oneスクライブ 清潔で磨かれた、両側</td></tr><tr><td> 170μmのカバーウエハ</td><td> SENSOR準備サービス</td><td> 7980 2Gウェーハ</td><td> Ø：100ミリメートル±0.5ミリメートル 厚さ：0.17ミリメートル±0.025ミリメートル 標準公差（記述のない限り） 表面仕上げ：SI：4-8A、SII：8-12A 干潟：OneプライマリSEMI-STD; Oneスクライブ 清潔で磨かれた、両側</td></tr><tr><td>酸化物の深い反応性イオンエッチング装置</td><td>アルバック</td><td>アルバックNL-6000 </td><td></td></tr><tr><td>アルゴンイオンレーザー</td><td>ケンブリッジレーザー研究所</td><td>レクセル95-SHG </td><td> λ= 258 nmの</td></tr><tr><td>アルゴンイオンレーザー</td><td>メレスグリオ</td><td></td><td> λ= 488 nmの</td></tr><tr><td>顕微鏡</td><td>オリンポス</td><td> IX-51</td><td></td></tr><tr><td>顕微鏡対物</td><td>トールラボ</td><td> OFR LMU-40X-UVB 0.5 NA </td><td> λ= 258 nmの</td></tr><tr><td>顕微鏡対物</td><td>オリンポス</td><td> UPLSAPO 60XW 1.2 NA </td><td> λ= 488 nmの</td></tr><tr><td> Nanopositioner </td><td>マッド·シティラボ</td><td>ナノLP100 </td><td></td></tr><tr><td>電動翻訳ステージ</td><td> Semprex株式会社</td><td> KL-シリーズの12から6436 </td><td></td></tr><tr><td> GaAsPのフォトンカウンター</td><td>浜松市</td><td> H7421-40 </td><td></td></tr><tr><td>モノクロ</td><td>ホリバ·インスツルメンツ株式会社</td><td> MicroHR </td><td></td></tr><tr><td> CCDカメラ</td><td>アンドールテクノロジー</td><td> IDUsの420A-BU </td><td></td></tr><tr><td>塩酸グアニジン（GuHCl） </td><td> Sigma-Aldrich社</td><tD&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

そのネイティブなコンフォメーションにによるタンパク質の折り畳み過程は、まだ、まだよくわかっていない生物学と人間の健康との関連性が高いです。この理由の1つは、折りたたみがタンパク質1に応じて、ナノ秒から秒以上に、スケールの広い範囲で行われます。従来のストップトフローミキサーは約1ミリ秒から始まるフォールディング動力学の測定を可能にしました。我々は最近〜8μsの2の変性剤は、〜100倍に希釈しマイクロミキサーを開発しました。ストップトフローミキサーとは異なり、このミキサーは発生しない乱流の層流領域で動作します。乱流の不在3-4実験するための優れた合意をミキサー内のすべてのフローの正確な数値シミュレーションを可能にします。 層流は、レイノルズ数のために再 ≤100を達成しています。水溶液の場合、これはミクロンスケールのジオメトリを必要とします。我々は、シリコンまたは縮合SILIとして、ハードディスク基板を使用（ 図1を参照）5-10μm幅10μmの深いチャネルを作るため、CA、。最小寸法は、混合領域の入り口に、サイズが1μmのオーダーである。チップは、光アクセスのための薄いガラスや石英ガラスカバーガラスで封入されている。典型的な総線形流量は 、Re〜10を得た〜1 m / sであるが、蛋白質の消費量は〜0.5 NL / sまたは1.8μL/ hrです。タンパク質濃度は、検出方法によって異なります。トリプトファン蛍光は典型的な濃度は100μM（1トリプトファン/蛋白質）であり、FRETのための典型的な濃度は、〜100nmである。 折りたたみ過程は、6 Mから0.06 M塩酸グアニジンに変性剤の急速な希釈によって開始されます。高変性剤の蛋白質は、中央のチャネルの下を流れ、変性剤（ 図2を参照してください）〜100倍も速く移動せずにバッファによって混合領域でどちら側でも満たされます。このジオメトリは、狭い中にタンパク質の流れの急速な収縮を引き起こすジェット〜100nmの広い。重いタンパク質分子の拡散が非常に遅いですが、光変性剤分子の拡散は1ミリ秒に1μm以下の拡散は、非常に急速である。蛋白質の周囲に変性剤の有効濃度を減少させる変性剤とタンパク質のストリームから変性剤の急速な希釈におけるタンパク質の結果の拡散定数の差。タンパク質のジェットは、走査型共焦点顕微鏡5を使用して観察することができる折り畳み中のタンパク質の観察チャネルおよび蛍光下に一定の速度で流れている。

Watch this Scientific Journal Video about Microfluidic Mixers for Studying Protein Folding at JoVE.com

Microfluidic Mixers for Studying Protein Folding

この研究では、〜8マイクロ秒で2つの溶液を混合することのできるマイクロミキサーの作製と使用について説明します。また、UV蛍光と蛍光共鳴エネルギー移動（FRET）を用いて分光検出とこれらのミキサの使用方法を示しています。

タンパク質の折り畳みを研究するためのマイクロ流体ミキサー

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

microfluidic-mixers-for-studying-protein-folding

Research

JoVE Journal

Bioengineering

15.0K ビュー.  Michigan State University. この研究では、〜8マイクロ秒で2つの溶液を混合することのできるマイクロミキサーの作製と使用について説明します。また、UV蛍光と蛍光共鳴エネルギー移動（FRET）を用いて分光検出とこれらのミキサの使用方法を示しています。

ビデオ: Microfluidic Mixers for Studying Protein Folding

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

単一粒子のためのマイクロ流体ベースの流体力学的トラップ

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

生物工学

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

蛍光分光法により測定膜タンパク質フォールディングの熱力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

マイクロ流体プラットフォームを使用した高スループットのタンパク質発現ジェネレータ

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

複数の異なった株を勉強するためのマイクロ流体デバイス

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMSサンプル導入のためのマイクロ流体チップ

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

タンパク質機能および抗体干渉によるタンパク質発現の変化の役割と三次元再構成を学びます

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全血からの超高感度のタンパク質検出のための全自動遠心マイクロ流体デバイス

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

シングルまたは共存化学/電気/せん断応力刺激下で細胞応答を研究するためのマイクロ流体デバイスを設計

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

バルブ付き多層マイクロ流体デバイスのための多段階可変高さのフォトリソグラフィ

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...