YGは、IDBからフェローシップでサポートされています。 INはアロンの仲間とエドモンドJ‭の教員仲間です。テルアビブ大学のバイオインフォマティクスのためのサフラ‬‭センター。‬この研究は、ISF助成​​金1499から1410によってサポートされていました。

1。スコッチテープマスターの作成​​5 <ol><li>紙の上で、拡大し、希望するマイクロチャネルのレイアウトを描画したり、プリントアウトする。我々のケースでは、設計は2つのY字型チャネル、ワイド各3ミリメートル（ 図2）から構成されています。 </li><li>スコッチテープの層を持つガラススライドをカバーしています。層の数は、チャネル（層当たり約60μm）の高さを決定します。私たちは3スコッチテープ層を使用していました。 </li><li>平らな面にレイアウト設計を置きます。デザインパターンオーバースライドの位置を合わせます。注意深くレイアウトに従ってメスでスライドガラス上にテープを切った。 </li><li>マイクロチャネルのレイアウトのものを除き、スライドガラスのすべての地域からセロハンテープを取り外します。 </li><li> 2〜3分間65℃で加熱オーブン中にスライドを入れます。エタノールで軽く清掃してください。 </li></ol> 2。 PDMSマイクロ流体デバイスを作製<ol><li>メーカーが推奨するように、PDMSの塩基及び硬化成分を混合する。私たちは、午前10時01分を使用硬化剤ベースの比率。約0.5cmの高さにペトリ皿にPDMSの混合物の約30ミリリットルを注ぐ。真空中のドガのPDMS必要に応じて。上向きにパターン化されたスコッチテープとPDMSに沈めスライドガラス。 PDMSの後にパターンを配置すると、スライドと皿の底部との間に気泡が形成されるのを防ぎます。皿は水準器を使って水平になっていることを確認しながら、65℃で48時間硬化させます。 </li><li>新しい90ミリメートルペトリ皿に深い約0.5mm層で、その結果、PDMSのミックスの3ミリリットルを注ぐ。 （スピンコーターが利用可能であれば、それはこのステージのために使用することができます）。 </li><li>ドガと2.1のように治す。 </li><li>そっとメスで希望のサイズにマイクロ流体デバイスを切り取る。この層は、 &quot;フロー層&quot;と呼ばれるでしょう。 </li><li>第二ペトリ皿から薄層PDMSの同じようなサイズの部分を切り取ります。これは、 &quot;井戸層&quot;と呼ばれるでしょう。 </li><li>流動層内の入口と出口のための穴を開けるために適切な大きさの生検パンチャーを（我々は、1.2 mmのIDを使用）を使用します。 </LI&gt; <li>レイアウト上のマイクロチャネルと整列して、2ミリメートル番号検パンチャーを使って、井戸のうち、設計レイアウトとパンチ以上厚いガラススライド上に井戸層を配置します。 </li><li>両方PDMS層だけでなく、エタノールで24ミリメートル×60 mmのカバーガラスを清掃してください。空気乾燥して清潔に保つ。 </li><li>プラズマはリバーシブルボンディング用の標準プラズマエッチャー（非可逆的結合用）またはハンドヘルドコロナ処理機6のいずれかを使用してカバーガラスや井戸層のPDMSの両方を扱う。慎重に（ 図3）密着性を引き起こすためにカバースリップの上に井戸層を配置。ゆっくり気泡を（必要に応じて）外にこする。 </li></ol> 3。細胞イメージング実験<ol><li>あなたはシリンジポンプで0.02の内径 &quot;（タイゴン）と柔軟なプラスチックチューブに接続されている適切なシリンジに準備ができて目的のメディアを持っていることを確認してください。 </li><li> Sを育てる液体YPDで所望の位相にセレビシエ株。徹底的にボルテックスと場所300μマイクロ遠心チューブに各株のL。 </li><li>優しくコンカナバリンA（シグマ）を各ウェルに2 mg / mlの1μlを置く。そっと水をピペッティングにより各ウェル倍から過剰コンカナバリンを洗い流す。 </li><li>コンカナバリン乾燥している間、SC培地欠けグルコース300μlの倍の株を洗ってください。細胞壁から残留グルコースを洗浄するコンカナバリンAに適切な遵守を支援します</li><li>徹底的にボルテックス細胞。ピペット0.75μlまたは独自のウェル（ 図3）にそれぞれの細胞懸濁液より少ない。優しく豊かな培地で残留細胞を洗い流す。次の2つの手順は、最大乾燥から細胞を防止するために、迅速に実行する必要があります。 </li><li>プラズマは直接井戸をぶつけないように注意しながら、徹底的に手持ちコロナ処理機を使用してチップや井戸を扱う。 2間のアライメントには細心の注意を払って、井戸にチップを慎重に置きます（このステップは、実体鏡下で行うことができます）。穏やかに、PDの2つの層を接着するために押すMS。徐々に気泡がチャネル内に残っていないことを確認しながら、約50μl豊富な培地でデバイスを完全に埋める。 </li><li>は、デバイスを接続し、適切な注入口にチューブを挿入し、メディアフローを開始します。これらは削除するのが難しいことができるように気泡がデバイスに導入されないように注意してください、これは入口または出口を切断することによって達成され、静かにカバーガラスを壊さないように注意しながら、気泡があるデバイスをタップすることができます。 </li><li>顕微鏡下に置き、各ウェルに適切な撮像ポイントを見つける。 </li><li>必要に応じて回復を可能にするために1時間以上のための豊富な培地流の下で細胞を維持。 </li><li>実験を開始します、我々はメディアの逆流を防止するために、2つのシリンジポンプのそれぞれから一定の流量を使用し、必要に応じて相対的な流量を変化させる。メディアチャネルの幅（ 図4）の80％以上が流れる中で0.8ミリリットル/時とポンプB 0.2〜200ml /時間の結果、ポンプを設定します。これは、動的にCすることができます2流量を変えることによって絞首刑。新しい条件は、チャネル2の全長に沿って数秒以内に安定させる。 </li></ol>

<ol>
	<li>Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+of+substrate-bound+laminin+orient+axonal+specification+of+neurons.">Gradients of substrate-bound laminin orient axonal specification of neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 12542-12547 (2002).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7165-7170 (2008).</li><li>Paliwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446, 46-51 (2007).</li><li>Bennett, M. R., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+measuring+gene+network+dynamics+in+single+cells.">Microfluidic devices for measuring gene network dynamics in single cells.</a> Nat. Rev. Genet. 10, 628-638 (2009).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Fabrication+of+Microfluidic+Devices+by+Replicating+Scotch-tape+Masters.">Simple Fabrication of Microfluidic Devices by Replicating Scotch-tape Masters.</a> Chips &#38; Tips. , (2010).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks.">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nat. Methods. 6, 71-74 (2009).</li></ol>

特別な利害関係は宣言されません。

本稿では、動的な条件の下で、次のいくつかの酵母菌株を同時に可能にするマイクロ流体デバイスを作成するためのシンプルな卓上法を提案する。一つのチャンネルに複数の系統に従うことにより、複数の系統で単一細胞応答の動態を比較するための信頼性の高いツールを提供しています。我々のアプローチの利点の一つは、クリーンルームを必要とせずに簡単なテクニックを使用してデバ?...

細胞は絶えず新陳代謝、転写プロファイルと細胞機能を変化させることにより、動的に変化する環境に反応している。これらの現象を研究するために、そのような変化を定量化することができる方法が必要とされる。そのような応答のための読み出しの一つのタイプは、栄養ストレスに応答して、ストレスに関連する転写因子の局在の変化である。 マイクロ流体デバイス1は、動的にセル2-4環境条件を操作するために使用されてきた。細胞の環境を正確に制御し、動的に変更することができます。細胞は外部条件の操作時も含めて、常時、ライブ画像化することが可能であり、最小限の試薬の量が必要とされています：彼らは、ライブセルイメージングのためのいくつかの利点を提示する。マイクロ流体デバイスのための非常にシンプルなデザインは、2つの条件の間に交替できるように、Y字型装置2です。我々は日常的にできるようにY字型のマイクロ流体デバイスを使用チャンネル内のセルを動的に変更したアプリケーション。我々は、酵母細胞内で蛍光標識されたストレス関連転写因子の局在の変化に追従するために、このデバイスを使用します。私たちは、栄養条件を変更するの下でこれらの変更を行います。たとえば、我々は非常に細かい時間分解能では、no-グルコース培地の期間内に媒体をグルコース含有のパルス（または一連のパルス）を提供することができる。しばしば、このような実験では、1つは、異なる細胞株（例えば、別の変異体、または異なる野生分離株）の応答を比較することに興味を持っています。 Y字型チャネルは、各チャネルに1株に限られている - 複数の菌株を使用する場合は、系統を区別する簡単な方法はありません。これを克服するために、異なるチャンネルを使用することができます。我々は複数の系統に同じ力学条件を適用したい場合、我々は、複数のチャネルとYチャネルの概念を組み合わせるしたいと思います。このソリューションを実装するには2つの課題があります：それはtを適用することは難しいことができます彼はすべてのチャンネルに同時に同じ条件、一つのデバイスに取り付けることができ、Yチャネル数に幾何学的な限界がある。そこで我々は、動的条件下で一つのチャンネルに複数の系統を表示するための方法を探していました。 ここでは、同じ動的条件下で1チャンネルでの撮像複数の株を対象とし、二層マイクロ流体デバイスを記述します。最下層は、穴の列に薄いPDMSから構成されています。この層は、我々はガラスにコンカナバリンAでそれらを付着し、異なる菌株を配置先となるウェルを作成するカバーガラスに装着されている。第二層はスコッチテープ法5を使用して作成されたY字型のマイクロ流体デバイスです。ウェル内で株を配置した後第二層は、迅速に整列され、最初の1に取り付けられた、異なる株が含まれている複数のウェルを有する一つのチャネルを作成します。この最終的なデバイスは、一つのチャンネルで複数の株に続いて、ここですべてのことができます株はちょうど同じ時間に同じ条件にさらされています。全体の設計と製造プロセスがないソフトリソグラフィと、簡単な手段を用いて、ベンチトップで行うことができます。

<table border="1"><tbody><tr><td>試薬/機器</td><td>会社</td><td>カタログ番号</td></tr><tr><td> PDMS-SYLGARD 184 </td><td>ダウコーニングの米国</td><td></td></tr><tr><td>減圧デシケーター</td><td>ナルゲン</td><td> 5310-0250 </td></tr><tr><td>生検パンチャー</td><td>テッド·ペラ社</td><td>ハリスユニコア15076（2ミリメートル）、15074（1.2ミリメートル） </td></tr><tr><td>シリンジポンプ</td><td> Chemyx </td><td>フュージョン200 </td></tr><tr><td>コロナ処理機</td><td>電気技術製品</td><td> BD-20 </td></tr><tr><td> Tygonチューブ</td><td>タイゴン</td><td> S-54-HL </td></tr><tr><td>コンカナバリンA- </td><td>シグマ</td><td> C7275 </td></tr><tr><td>スコッチテープ</td><td> 3Mスコッチ</td><td>透明テープ1/2 &quot; </td></tr></tbody></table>

a microfluidic device for studying multiple distinct strains

環境変化への細胞応答の研究は、多くの実験的な課題があります。セルは、比較の方法で、多くの場合、変化する条件の下で撮像する必要があります。マルチウェルプレートは、日常的に多くの異なる株または細胞株を比較するために使用されるが、環境動態の制御は制限付きで許可されています。マイクロ流体デバイスは、他の一方で、周囲の条件で絶妙な動的な制御を可能にするが、それはイメージに挑戦し、それらのいくつかの菌株よりも区別されています。ここでは、簡単かつ迅速に一つのチャンネルに複数の異なる酵母菌株に動的に変化する条件を適用することができるマイクロ流体デバイスを製造する方法について述べる。デバイスは、ソフトリソグラフィーを必要とせずに簡単な手段によって設計され、製造されています。それは、マイクロウェルを宿す第二層に付着したY字型の流路で構成されています。菌株は、独立したマイクロウェル内に置かれ、まったく同じ動的条件下で結像される。我々は、デモ異なる酵母菌株の栄養変化のパルスにタンパク質局在応答を測定するためのデバイスの使用をnstrate。

異なる系統間の分離が可能であることを示すために、我々は代替ウェルで2つの区別酵母株をイメージした。イメージングフル井戸は井戸（ 図5a、b）の間には、セルのリークを示していない。両株はYFPでタグ付けされた転写因子MSN2を持っています。動的に条件を変えての同時効果をテストするために、我々は核 ​​（ 図5c、d）へMsn2-YFPの局在をもたらし無グルコー?...

Watch this Scientific Journal Video about A Microfluidic Device for Studying Multiple Distinct Strains at JoVE.com

A Microfluidic Device for Studying Multiple Distinct Strains

私たちは、クリーンルームやソフトリソグラフィーを必要とせずに、複数の異なる株に類似した、動的な条件を適用することができるマイクロ流体デバイスを製造するための簡単​​な方法を提示する。

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-device-for-studying-multiple-distinct-strains

Research

JoVE Journal

Bioengineering

8.7K ビュー.  Tel Aviv University. 私たちは、クリーンルームやソフトリソグラフィーを必要とせずに、複数の異なる株に類似した、動的な条件を適用することができるマイクロ流体デバイスを製造するための簡単​​な方法を提示する。

ビデオ: A Microfluidic Device for Studying Multiple Distinct Strains

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

腫瘍微小環境を再作成するためのマイクロ流体デバイス<em&gt;体外で</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

生物工学

Microfluidic Mixers for Studying Protein Folding

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 for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

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 ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

マイクロピラーとマイクロ流体デバイス内のバイオフィルムストリーマ形成のためのプロトコル

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

マイクロスケールシュリーレン法によるマイクロ流体デバイス内の混合不均一性の分析

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

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 ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

ガットオンチップマイクロ流体デバイスにおけるマイクロ加工ヒト腸絨毛と生活Microbiomeの共培養

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

ドライ フィルムのレジストを用いた電気化学マイクロ流体バイオ プラットフォーム: デバイス作製、チップの試金の準備、およびシステム操作

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

マイクロ流体デバイスにおける組織モデルと perfusable の血管ネットワーク

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

ハイスループットマイクロ流体装置を用いた動的環境条件の生成

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

コンビナトリアルプラグ製造のためのマイクロ流体デバイスの構築

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

著者スポットライト:Precision Oncologyにおける計算アプローチと実験的アプローチの統合

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

コンビナトリアルプラグ製造のための二層マイクロ流体デバイス

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...