この作品は、化学科からと研究局（RCAP 13から8032）からの内部サポートを含め、科学と工学のウェスタンケンタッキー大学のオグデン大学によってサポートされています。 WKU顕微鏡施設でドクター・ジョンAndersland（SEM像）と、燃焼科学と工学（BET分析）のためWKU研究所の准教授ヤン曹操の支援は、この作業を行うの中心となっています。

エマルジョンの調製<ol><li>エマルジョンの内容<ol><li>質量は、塩の適切な量は、0.03-M溶液10mlを作製しました。プラチナ、亜鉛（II）クロリド（のZnCl 2）尺度0.032 g及び塩化ナトリウム（NaCl）で測定値0.018 gで（IV）、塩化尺度0.101 gでした。 </li><li>個々の試験管において、脱イオン水10ml中に各塩を溶解します。後で使用するために脇に置きます。 </li><li>この手順全体の内容のために20ミリリットル、密封可能なガラスバイアルを使用します。ガラスバイアルに風袋引き。 </li><li>ゆっくりと攪拌棒を越えてゼロ化された規模で休んでガラスバイアルにそれを注ぐことによって、ビニル末端ポリジメチルシロキサンを計量。 （1.080ミリリットルに相当）1.02グラムを計量。 注：このポリ​​マーの高粘度は、ピペッティングは非現実的になります。 </li><li>ピペットバイアルにn-ヘプタンの1.02ミリリットル。バイアルに非イオン性界面活性剤（モノオレイン酸ソルビタン）の2滴を追加します。ピペット塩溶液の0.3ミリリットルバイアルにDI水の0.45ミリリットル。 </li><li>しっかりと蓋をネジ止めすることにより、ガラスバイアルを密封。超音波処理を開始する前に、エマルジョンを開始するために60秒間激しく振ります。 </li></ol></li><li>水浴超音波処理装置の構成<ol><li>最小充填ラインまで水で超音波処理器を埋めます。 400ミリリットルのビーカーに水道水250ミリリットルを追加します。水位がちょうど縁にあるように氷で400ミリリットルのビーカーを埋めます。 </li><li>水浴ソニケーター内でこのビーカーを置きます。必要に応じて調整し、超音波処理器に充填ラインを確認してください。リングは水浴超音波処理のすぐ横に立って置きます。 </li><li>アームが引き出されているように、2つのリングは、クランプをスタンドにそれらを配置し使用して、リングに垂直に立って、それが氷の水で満たされたビーカーに下向きとなるように別のものを水槽に向けて延長されます。リングに別のクランプを取り付け、温度が超音波処理を通してモニターすることができるように400ミリリットルのビーカーにダウン温度計を立ちます。 </li></ol></li><li> Emulsification手順<ol><li>それは完全にビーカーにダウン突出クランプにそれを固定することによって、400 mlのビーカーに沈められるようにエマルジョンを含むバイアルを置きます。 </li><li> PDMSの混合物を含有するガラスバイアルは摩擦による熱を除去するために、ビーカーの側面に触れていないことを確認してください。 </li><li>超音波処理装置の電源をオンにし、7分間の超音波処理時間を設定します。エマルジョンは敏感非常に熱であるため、ビーカー内の温度は、超音波処理を通じて、0〜5℃の間であることを確認してください。ビーカー内の温度が望ましいたら、超音波処理を開始します。 </li><li>超音波処理の7分後、バイアルを取り外し、ゆっくりとエマルジョン中に形成する可能性の塊を除去するために、バイアルの上部を保持し、1分間/スワールを振ります。 </li><li>ビーカーの内容物を廃棄してください。 250mlの水を補充し、ビーカーの上部CM 1内に充填するために氷を追加します。 </li><li>バッククランプでバイアルを置きます。 RESUMに氷水下でそれを沈めます7分の電子の超音波処理。 </li><li>繰り返しは、8の合計1.3.6に7分間超音波処理期間を1.3.3ステップ、または超音波処理は、均質であることが表示され、何の塊が存在しなくなるまで。室温で保管してください。 注：乳化は数日間安定であるべきであり、それが均質に表示されるまで、7分間隔で、上記のように超音波処理することによって回収することができます。室温で保管してください。 </li></ol></li></ol> 2.架橋<ol><li>トリエトキシシラン/界面活性剤溶液を添加するためのセットアップ<ol><li>試験管に5.4ミリリットルトリエトキシシランをピペット。後で使用するためにボンネットの下に試験管ラックに配置します。 </li><li>氷水で400ミリリットルのビーカーを記入し、ボンネットの下に置きます。次にリングを取り付けクランプでスタンド400ミリリットルのビーカーの場所に、ビーカーの開口部の真上に拡張しました。これは、トリエトキシシランの添加のための氷浴になります。 </li><li>リングスタンドの反対側にホットプレートを置きます。約700ミリリットルで800ミリリットルのビーカーを埋めます水道水。ホットプレートの上に置きます。 </li><li>ホットプレートの電源をオンにし、800ミリリットルのビーカーの内側75〜85℃の温度を維持します。 800ミリリットルのビーカー内の水の温度を監視することができるようにリングスタンドに温度計をクランプを取り付けます。 </li><li>水の375ミリリットル（4.62ミリモル）にドデシル硫酸ナトリウムを0.5gを溶解させて界面活性剤溶液を生成します。きれいな、空の試験管に界面活性剤溶液の約10ミリリットルを追加します。 </li><li>界面活性剤の試験管にボンネットの下にリングスタンドに別のクランプを取り付けすると、その液面が800ミリリットルビーカー内の水の表面の下になるように固定されています。熱平衡のために10分を許可します。 </li><li>濾紙を湿らせ、小さな漏斗の上部に配置します。 250mlの三角フラスコ内の漏斗の茎を置き、ボンネットの下にフラスコを置きます。 </li></ol></li><li>トリエトキシシランを添加します<ol><li>氷水のくちばしの上にクランプでエマルジョンバイアルを置きますフード内側えー。バイアル内容物は、水の表面の下になるようにクランプでPDMS混合物を置きます。トリエトキシシランの添加は発熱反応を起こすので、エマルジョンは、その構造を維持するために、冷保たなければなりません。 </li><li>気体の蓄積を回避するために、ガラスバイアルから蓋を外します。 </li><li>ゆっくりと約10秒（約0.5ミリリットル/秒）の期間にわたって連続的な流れでガラスバイアルにトリエトキシシランを含有する試験管を注ぎます。 注意：トリエトキシシランの添加は発熱反応と苛性塩化水素（HCl）ガスの放出を開始します。バイアルは、非常に高温になり、有毒ガスが混合物から進化していきます。トリエトキシシランを添加しながら、内容を攪拌しないでください。 </li><li>熱保護手袋を着用したまま完全にトリエトキシシランを添加した後、ゆっくりとガラス撹拌棒で中身をかき混ぜます。 2分間またはバイアルから進化したガスが停止するまで待ちます。 </li><li> Foll架橋のため、試料中の目に見える相分離は存在しません。塊が存在している場合は、バイアルを密封し、蓋によってバイアルを保持しながら20秒間厳密に振ります。 </li></ol></li><li>ビーズ生産<ol><li>ガラス瓶から架橋されたエマルジョンを描くようにきれいな、ガラスパスツールピペットを使用してください。液滴間でできるだけ時間をかけ試験管に架橋されたエマルションの滴下界面活性剤溶液に（75および85℃の間に維持すべきである）を加えます。 </li><li> 1分30秒のエマルジョンを添加した後、界面活性剤溶液をゆっくりと固体が試験管の内部に形成され始めるようにガスを発生し始めます。 </li><li>熱保護手袋を装着したまま、クランプのうち、試験管を取り、ボンネットの下に濾過装置にその内容全体を注ぎます。 5分間フィルター。フィルターからろ紙を取り除きます。 </li><li> Hの下でO / Nの乾燥のために時計のガラス、別のビーズ上に濾過した固体を転送OOD。ビーズの洗浄は、無期限に延期することができます。ストアは、必要になるまで密封したガラスバイアル中で、室温でビーズを乾燥させ、きれいな、使用直前に。 </li></ol></li><li>ビーズクリーニング<ol><li>ボンネットの下に別の濾過装置を作成し、ろ紙の上に漏斗内部乾燥ビーズを配置します。 </li><li>すべてのビーズがすすぎされることを保証するために、わずかにそれらを動き回る、優しくビーズを洗浄するために脱イオン水で満たされたプラスチック製の洗浄ボトルを使用してください。 </li><li>ビーズはフードの下時計皿の上にそれらを配置することによって、1時間乾燥させます。水ですすぐために同じ方法を用いてビーズを洗浄するためにヘキサンを充填した洗浄ボトルを使用してください。 </li><li>時計皿の上にビーズを配置します。乾燥するためにボンネットの下に時計のガラスビーズを置きます。 </li><li>ビーズが完全に乾燥した後、将来の使用のために室温で小さな密閉可能なガラスバイアルとストアに配置します。 </li></ol></li><li> SEM分析とSEMの設定のためのビーズを取り付け<ol><li> DOのストリップを配置しますビーズがマウントされる上に、スタブの上にUBLE両面カーボン導電性テープ。はさみを使用して、エッジの上に何のテープハングを保証しないようにスタブを中心にトリミングします。 </li><li>平らな表面上のスタブの下に濾紙を置きます。接着剤下面が露出するようにテープから最上層を削除します。 </li><li>ゆっくりスタブの上にビーズを注ぎます。いくつかのビーズは、テープに固執するが、ほとんどは跳ね返るとろ紙上に着陸する予定。彼らはろ紙上にとどまった場合、バイアルにこれらのバックを注ぎます。必要に応じて汚染される（2.4.5を2.4.2による）任意のビーズを洗浄、繰り返します。 </li><li>ビーズは、スタブ上で固定されていることを保証するために、電球の注射器を使用し、軽くスタブ表面に非常に密接に吹きます。わずか数がテープに付着した場合は、スタブ上でより多くのビーズを注ぎます。すべてのビーズをSEM室にスタブを配置し、それを排出する前に、安全であることを確認してください。 </li><li>サンプルが正常にマウントされた後、彼らは今のSEM肛門を受ける準備ができていますysis 15。ビーズの表面の特徴の解像度を最適化するために、15 keVの時にLOW VACモードで画像を収集します。 </li></ol></li></ol>

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The authors have nothing to disclose.

図1の他のSEM画像を図1（a）の比較によって分かるように、このプロトコル（および電解質濃度及びアイデンティティを調節することによって）を用いて製造ビーズは、低イオン強度のエマルジ ​​ョンで製造されたものとは根本的に異なっている。使用する我々の最初の報告さらに水-脂肪族インターフェース 14で重合架橋を触媒することを意図してのPtCl <s...

Polydimethylsiloxane (PDMS) is one of the most widely used silicone compounds. Its biocompatibility has led to widespread use in implant and other biomedical engineering structures1,2. It is trivially cross-linked into elastic structures using an organosilyl compound (such as triethoxysilane), a simple and reliable procedure which has made it useful for cast polymer applications where some flexibility is required3. Once cross-linked, PDMS is largely inert, particularly in biological conditions, and is therefore useful for a variety of food and medical applications4,5. Ease of casting, chemical inertness, and hydrophobicity have made it a natural choice for microfluidic devices6,7. Its affinity for non-halogenated, non-polar organic compounds has made it a popular stationary phase in separations chemistry8-10. 
Recently, microbubble fabrications have been used to generate porous beads for use as catalyst structural substrates or in chemical separations11,12. In both applications, ideal materials will have a maximized surface-area-to-volume (SAV) ratio for best efficiency. In a microbubble fabrication process, microstructuring of materials is typically accomplished by isolating the polymer in aliphatic &#8220;microbubbles&#8221; by emulsification in an aqueous continuous phase. The initial report of microporous PDMS beads produced them by mechanical emulsification of two phases (aliphatic and aqueous)13. The stock PDMS liquid (and its cross-linking agent) is dissolved into the aliphatic phase, which is structured into microscopic beads by being forced to cavitate within the (continuous) aqueous phase. The emulsification is stabilized by the addition of a non-ionic surfactant. When the emulsion is added dropwise to a heated bath, solid beads form by agglomeration of the microbubbles into clusters of tiny spheres of cross-linked PDMS. Our goal in this protocol is to modify this procedure to develop beads with an inverted porosity to improve the SAV ratio of the material.
As reported previously, control of the beads can be directed to some extent by the aliphatic:aqueous ratios in the emulsion. However, we have reported recently that addition of platinum(IV) chloride (PtCl4) inverts the porosity: materials are formed in which the PDMS is riddled with concave pores14. This indicates that the aqueous layer cavitates inside the aliphatic one, despite having similar aliphatic:aqueous ratios to those published in the original work13. The primary advantage of our method is that this concave porosity should naturally result in an increased SAV ratio, and thus, improved efficiency for analytical chemistry applications. While we are continuing to explore the specific effects of the addition of the platinum compound, we show here that the same effect can be accomplished using any aqueously soluble ionic compound, though perhaps to a reduced extent. Because our techniques also differ in some key aspects from what has been previously reported, we present our protocols here as a video to encourage others to extend our methods. Most notably, we use a common bath sonicator of the type used to clean glassware or other equipment, rather than the (considerably more expensive) probe sonicator often used in microbubble fabrication. This adjusted approach to the microbubble fabrication procedure could potentially be extended for the production of large quantities of bulk materials as well, creating porous sheets or slabs which could have applications for biomedical devices, aerospace and automotive industry, or substrates for chemical catalysis. Users seeking to generate high-SAV-ratio, microstructured materials using other similar polymers for such analyses may find that our protocols can be extended to any polymer for which the microbubble emulsion technique can be applied.

<table><tbody><tr><td>Poly(dimethylsiloxane), vinyl terminated</td><td>Sigma-Aldrich</td><td>68083-19-2</td><td></td></tr><tr><td>n-Heptane</td><td>Sigma-Aldrich</td><td>142-82-5</td><td>Flammable</td></tr><tr><td>Triethoxysilane</td><td>Sigma-Aldrich</td><td>998-30-1</td><td>Flammable, Accutely Toxic</td></tr><tr><td>Sorbitan Monoleate (Span-80)</td><td>Fluker</td><td>1338-43-8</td><td></td></tr><tr><td>Platinum(IV) Chloride</td><td>Sigma-Aldrich</td><td>13454-96-1</td><td>Accutely Toxic</td></tr><tr><td>Zinc(II) Chloride</td><td>Sigma-Aldrich</td><td>7646-85-7</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>2.8 L Water Bath Sonicator</td><td>VWR</td><td>97043-964</td><td></td></tr></tbody></table>

microbubble fabrication of concave-porosity pdms beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

異なる電解条件エマルジ ​​ョンから生じるビーズの代表的なSEM像を図1に示す 。 図1Aは DuFaudしたもの、 ら 13に類似したビーズを示し、任意の電解質を添加せずに、我々の手順を用いて製造。ビーズは、各金属イオンのための別の形態で得られ、 図1B-Dに示します。示された全ての画像については、0.03-Mの電解質溶液の300μl?...

Watch this Scientific Journal Video about Microbubble Fabrication of Concave-porosity PDMS Beads at JoVE.com

Microbubble Fabrication of Concave-porosity PDMS Beads

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

凹面気孔率PDMSビーズのマイクロバブル作製

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

microbubble-fabrication-of-concave-porosity-pdms-beads

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.3K ビュー.  Western Kentucky University. Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized. 

ビデオ: Microbubble Fabrication of Concave-porosity PDMS Beads

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

ポリマー基板上へのマイクロ·サブミクロンのパターンを生成するためのリソグラフィMicropunching

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

工学

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

生物学

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

エラストマーのマイクロバルブの配列で制御マイクロ流体チップ

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

ハイスループットマイクロカプセル化シングルセルと複数セル

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

生物工学

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

コロイド粒子の自己組織化プロセスを介して、PDMSマイクロ流体チップ内のサブ50 Nmのナノ流体ジャンクショ​​ンを作成します

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

マイクロ流体技術は、Cell変形能の探査

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

生体の屈折率-屈折率整合素子の作製

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

ポリジメチルシロキサン（PDMS）同軸フローフォーカス・デバイスを使用したダブルエマルションの生成

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

多孔質媒体中の微生物トランスポート研究のためのシンプルなマイクロ流体システム：マイクロワールドでのウィンドウ

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

凹面気孔率PDMSビーズのマイクロバブル作製

要約

さらに動画を探す

ポリマー基板上へのマイクロ·サブミクロンのパターンを生成するためのリソグラフィMicropunching

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

エラストマーのマイクロバルブの配列で制御マイクロ流体チップ

ハイスループットマイクロカプセル化シングルセルと複数セル

コロイド粒子の自己組織化プロセスを介して、PDMSマイクロ流体チップ内のサブ50 Nmのナノ流体ジャンクションを作成します

マイクロ流体技術は、Cell変形能の探査

生体の屈折率-屈折率整合素子の作製

ポリジメチルシロキサン（PDMS）同軸フローフォーカス・デバイスを使用したダブルエマルションの生成

多孔質媒体中の微生物トランスポート研究のためのシンプルなマイクロ流体システム：マイクロワールドでのウィンドウ

ペアになっているビーズと磁石配列変数の凹面形状とマイクロウェルを成形

凹面気孔率PDMSビーズのマイクロバブル作製

要約

さらに動画を探す

ポリマー基板上へのマイクロ·サブミクロンのパターンを生成するためのリソグラフィMicropunching

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

エラストマーのマイクロバルブの配列で制御マイクロ流体チップ

ハイスループットマイクロカプセル化シングルセルと複数セル

コロイド粒子の自己組織化プロセスを介して、PDMSマイクロ流体チップ内のサブ50 Nmのナノ流体ジャンクショ​​ンを作成します

マイクロ流体技術は、Cell変形能の探査

生体の屈折率-屈折率整合素子の作製

ポリジメチルシロキサン（PDMS）同軸フローフォーカス・デバイスを使用したダブルエマルションの生成

多孔質媒体中の微生物トランスポート研究のためのシンプルなマイクロ流体システム：マイクロワールドでのウィンドウ

ペアになっているビーズと磁石配列変数の凹面形状とマイクロウェルを成形

コロイド粒子の自己組織化プロセスを介して、PDMSマイクロ流体チップ内のサブ50 Nmのナノ流体ジャンクションを作成します