EPSRC、HFSP、韓国の国立研究財団（助成番号R11 - 2009 - 044 - 1002 - 0K20904000004 - 09A050000410）：著者は、以下の研究会と助成金の支援を感謝したい。

<ol>
	<li>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=The+origins+and+the+future+of+microfluidics.">The origins and the future of microfluidics.</a> Nature. 442, 368-368 (2006).</li><li>Zheng, B., Angew, I. s. m. a. g. i. l. o. v., F, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microfluidic+Approach+for+Screening+Submicroliter+Volumes+against+Multiple+Reagents+by+Using+Preformed+Arrays+of+Nanoliter+Plugs+in+a+Three-Phase+Liquid/Liquid/Gas+Flow.">A Microfluidic Approach for Screening Submicroliter Volumes against Multiple Reagents by Using Preformed Arrays of Nanoliter Plugs in a Three-Phase Liquid/Liquid/Gas Flow.</a> Chem. Int. Ed. 44, 2520-2520 (2005).</li><li>Huebner, A., Sharma, S., Srisa-Art, M., Hollfelder, F., Edel, J. B., Demello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdroplets:+A+sea+of+applications.">Microdroplets: A sea of applications.</a> Lab on a Chip. 8, 1244-1244 (2008).</li><li>deMello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+detection+of+chemical+reactions+in+microfluidic+systems.">Control and detection of chemical reactions in microfluidic systems.</a> Nature. 442, 394-394 (2006).</li><li>Thorsen, T., Roberts, R. W., Arnold, F. H., 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=Dynamic+pattern+formation+in+a+vesicle-generating+microfluidic+device.">Dynamic pattern formation in a vesicle-generating microfluidic device.</a> Phys. Rev. Lett. 86, 4163-4163 (2001).</li><li>Anna, S. L., Bontoux, N., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+dispersions+using+"flow+focusing"+in+microchannels.">Formation of dispersions using "flow focusing" in microchannels.</a> Appl. Phys. Lett. 82, 364-364 (2003).</li><li>Casadevall i Solvas, X., Srisa-Art, M., deMello, A. J., Edel, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+Fluidic+Mixing+in+Microdroplets+with+1+&#x03BC;s+Time+Resolution+Using+Fluorescence+Lifetime+Imaging.">Mapping of Fluidic Mixing in Microdroplets with 1 &#x03BC;s Time Resolution Using Fluorescence Lifetime Imaging.</a> Anal. Chem. 82, 3950-3950 (2010).</li><li>Robinson, T. A., Schaerli, Y., Wootton, R., Hollfelder, F., Dunsby, C., Baldwin, G., Neil, M., French, P., deMello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+background+signals+from+fluorescence+thermometry+measurements+in+PDMS+microchannels+using+fluorescence+lifetime+imaging.">Removal of background signals from fluorescence thermometry measurements in PDMS microchannels using fluorescence lifetime imaging.</a> Lab. Chip. 9, 3437-3441 (2009).</li><li>Huebner, A., Srisa-Art, M., Holt, D., Abell, C., Hollfelder, F., deMello, A. J., Edel, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+protein+expression+in+single+cells+using+droplet+microfluidics.">Quantitative detection of protein expression in single cells using droplet microfluidics.</a> Chem. Commun. 1218, (2007).</li><li>Edel, J. B., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+single+molecule+FRET+efficiency+determination+for+surface+immobilized+DNA+using+maximum+likelihood+calculated+lifetimes.">Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood calculated lifetimes.</a> J. Phys. Chem. B. 73, 2986-2990 (2007).</li></ol>

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我々は、マイクロ流体デバイスの製造、液滴の形成と試薬のカプセル化と、処理液滴の光学的に検出するための実験のセットアップと関連プロトコルを記述している。 （液滴内の単一細胞の検出と流れる滴内部の混合プロセスのマッピング）を選択した二つの例は、現在、液滴のマイクロフルイディクスを検討されている一般的なアプリケーションを表します。 提示の実験が示すように、液滴マイクロ流体プラットフォームの使用は、しかし、そのような高スループット、準最適な閉じ込め、高い再現性...生産、大量の情報およびこの情報が作成されている時に高速などの特定の魅力的な特性を、提示完全な利点は、これらの小型のプラットフォームから得られるかどうか、高時空間分解能を持つ高速な検出方法を使用する必要があります。このケースではこれがであることを示している高精度の蛍光分光技術を使用して可能。特に、FLIMの検出技術は、（数nsと短い繰り返し周期とパルスレーザーを利用する）急速に流れる滴（1秒間に200前後の）から、非常に高速な情報を取得する能力を明らかにする。この場合、検出されたイベントの時間分解能は1μsと低かった。液滴の大きさと濃度の限界の面では、より大きな開口数のレンズと大きな出力レーザーの使用が容易に5μmの（液滴の大きさの制限は、フォトリソグラフィの解像度によって課されているような小さな液滴内nMの濃度の検出を可能にするでしょうサブミクロンの位置決めステージの製造プロセスと）。 マイクロ流体の液滴は下に単一のターゲット/イベントに、試薬の少量を含む大規模な実験を実施するために理想的な候補です。この技術の現在の制限は、DRを生成するために困難を伴うハイスループットな方法でさまざまなソースの大規模な様々な（数十または数百）からoplets。さらに、生成された各単一液滴をターゲットにして操作する難しさは、これらの技術の実用性には厳しい制限を課している。したがって、これらの問題は、マイクロ流体液滴のプラットフォームは、高スループットのアプリケーションの研究と分析の標準的な参照方法になることを可能にする必要な技術的ソリューションの開発を目指した重要な研究努力の中心になります。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>フルオレセイン5 - イソチオシアネート（FITC） </td><td>試薬</td><td>シグマアルドリッチ</td><td> F3651 </td></tr><tr><td>ストレプトアビジン-のAlexa Fluor ® 488 </td><td>試薬</td><td>分子プローブ</td><td> S11223 </td></tr><tr><td> Perfluorodecalin </td><td>試薬</td><td>シグマアルドリッチ</td><td> P9900 </td></tr><tr><td> 1H、1H、2H、2H - perfluorooctanol </td><td>試薬</td><td>シグマアルドリッチ</td><td> 370533 </td></tr><tr><td> Sylgard 184シリコーンエラストマーキット</td><td>試薬</td><td>プレミアファーネル英国LTD </td><td> 1673921 </td></tr><tr><td>オーラミン- O </td><td>試薬</td><td>シグマアルドリッチ</td><td> 861030 </td></tr><tr><td> 485 nmのパルスダイオードレーザー</td><td>機器の</td><td> PicoQuant </td><td> LDH - PC - 485 </td></tr><tr><td> TCSPCカード</td><td>機器の</td><td> PicoQuant </td><td> TimeHarp 100 </td></tr><tr><td>ダイクロイックミラー</td><td>機器の</td><td>クロマテクノロジー株式会社</td><td> z488rdc、 </td></tr><tr><td>顕微鏡の目的</td><td>機器の</td><td>オリンポス</td><td> UPLSAPO 60XW </td></tr><tr><td>アバランシェフォトダイオード</td><td>機器の</td><td> AQR - 141、EG＆G、パーキンエルマー社製）。 </td><td> AQR - 141 </td></tr><tr><td> DMEM </td><td>試薬</td><td>インビトロジェン</td><td> ABCD1234 </td></tr><tr><td> SYTO9 </td><td>試薬</td><td>インビトロジェン</td><td> S34854 </td></tr><tr><td>ヨウ化プロピジウム</td><td>試薬</td><td>インビトロジェン</td><td> P3566 </td></tr><tr><td>大腸菌上位10菌株</td><td>細胞</td><td>インビトロジェン</td><td> C4040 - 10 </td></tr></tbody></table>

fluorescence detection methods for microfluidic droplet platforms

化学と生物学を行うためのマイクロ流体プラットフォームの開発は、大部分のシステムの小型化に伴う潜在的な利益の範囲によって駆動されています。利点は、効率的にサンプルのfemotoリットルのボリューム、機能コンポーネントの容易な統合、大規模な多重化、強化された分析のスループット、改良されたコントロールと減少楽器の足跡に向けた本質的な素因ナノに処理する能力が含まれています1。 近年では多くの関心は、液滴ベース（またはセグメント化されたフロー）マイクロ流体システムとハイスループット実験のプラットフォームとしての可能性の開発に焦点を当てている。ここでは2-4、油中水型のエマルジ ​​ョンが自発的にマイクロ流体チャネルで形成するように作られて2つの不混和相の間のキャピラリー不安定の結果として。重要なのは、正確に定義されたボリュームや組成の微小液滴は、周波数で発生させることができます数kHzの。さらに、連続的な不混和相、サンプルのクロストークの両方と分散（拡散およびテイラーベース）で区切られた、分離区画内で関心の試薬をカプセル化することによって除去することができる、これは最小限のクロスコンタミネーションと時間分析プロセスをする能力につながる偉大な精度で。さらに、液滴の内容とチャネル壁（連続相によって湿潤されている）の間には接点がないので、チャネル壁上に試薬の吸収と損失が防止される。 この種の液滴が生成され、処理された後は、必要な分析情報を抽出することが必要です。この点では選択肢の検出方法、検出の高感度と低制限を提供する、迅速でなければならない、分子種の範囲に適用可能である、非破壊的であると安易な方法で、マイクロ流体デバイスと統合することができるようになります。このニーズに応えるために、我々はとして開発しました。実験的なツールと小音量の環境からの光物理大量の情報の抽出を可能にし、物理的、化学的、生物学的パラメータの広い範囲の分析に適用可能なプロトコルのuite。ここにこれらのメソッドの二つの例を提示し、単一細胞とピコリットル容量の液滴内部の混合プロセスのマッピングの検出に適用されます。我々は、マイクロ流体チップ製造、光学セットアップおよび液滴の生成と検出のプロセスを含む全体の実験的なプロセスを報告する。

Watch this Scientific Journal Video about Fluorescence detection methods for microfluidic droplet platforms at JoVE.com

Fluorescence detection methods for microfluidic droplet platforms

彼らはkHzのレートで安価にピコリットル、自己区分血管を生成することができるので、液滴ベースのマイクロ流体プラットフォームは、ハイスループットの実験のための有望な候補である。 、高速で高感度かつ高分解能蛍光分光学的手法との統合により、これらのシステム内で発生する大量の情報を効率的に、抽出活かされ、利用することができます。

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 ...

fluorescence-detection-methods-for-microfluidic-droplet-platforms

Research

JoVE Journal

Bioengineering

21.9K ビュー.  Imperial College London. 彼らはkHzのレートで安価にピコリットル、自己区分血管を生成することができるので、液滴ベースのマイクロ流体プラットフォームは、ハイスループットの実験のための有望な候補である。 、高速で高感度かつ高分解能蛍光分光学的手法との統合により、これらのシステム内で発生する大量の情報を効率的に、抽出活かされ、利用することができます。

ビデオ: Fluorescence detection methods for microfluidic droplet platforms

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

生物工学

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

ハイスループットマイクロ流体の迅速かつ低コストのプロトタイピング包装方法

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

細胞内小胞の追跡への応用：変形二光子顕微鏡での3d軌道の追跡

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

商業顕微鏡でのライブ細胞膜上の分子拡散法への高速蛍光イメージングから

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

リン脂質単層の二次元拡散を測定するためにドロップレットをマージした後蛍光回復

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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 ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

粒子の空間追跡のための多重化された電子の検出とマイクロ流体プラットフォーム

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.