This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1.設計とMDFチップの作製<ol><li> AutoCADなどの市販のソフトウェアを使用して、個々のアクリル層パターンを描画し、パターンを保存します。 <ol><li> 図1Aの 4層アクリル板パターンの設計を見直し、層間接続を確認してください。 </li></ol></li><li> CO 2レーザースクライバー（ 図1B、 図2A、 および図2B）を用いたレーザアブレーションによってすべてのアクリルシートと両面テープを製造する。16 <ol><li>レーザースクライバーをオンにして制御するラップトップにマシンを接続します。 </li><li>商用ソフトウェアを実行し、クリックすることで、ステップ1.1で設計されたパターンを開く」設計パターンファイルを。」 </li><li>レーザースクライバーのXYZステージ上の空白のアクリル板や両面テープ片を置きます。 </li><li>自動alignmenアクリルシート又は両面テープの表面上にレーザービームの焦点を設定しますレーザースクライバーの製造元から提供されているTスティック。 </li><li>アクリルシートや両面テープを直接加工用レーザースクライバーに設計されたパターンを送信します。 </li></ol></li><li>ピンセットを用いてアクリルシートの保護紙を取り外し、窒素ガスで清浄な表面を吹きます。 </li><li>アクリルシートを積層し、110で45分間熱ボンダーで2キロ/ cm 2の圧力下で一緒に結合 フロー/電気刺激チャネル組立体を形成する°C。 </li><li>チップ内の細胞培養基質として顕微鏡カバーガラスを準備します。 <ol><li>染色ジャーにカバーガラスを入れ、材料リストに記載されている洗剤の10倍希釈で瓶を埋めます。 </li><li>超音波steri用洗剤にカバーガラスや染色ジャーを入れ、15分間カバーガラスを清掃してください。 </li><li>染色ジャーの外に希釈した洗剤を注ぎ、蒸留水で瓶を再充填し、ステップ1.5を繰り返します。2〜3回。 </li><li>両面テープに接着する前に、窒素ガスと一緒に吹き付けて清掃カバーガラスを乾燥させます。 </li></ol></li><li>ステップ1.2でパターニング両面テープと流れ/電気刺激チャネルアセンブリに洗浄カバーガラスを接着。 </li><li>スーパー接着剤でMDFチップアセンブリのレイヤ1の個々の開口部にアクリルアダプタの13枚を接着。 MDFチップアセンブリは、完全な（ 図2D）です。 </li><li>使用前にUV照射の30分で完全なMDFチップアセンブリを滅菌します。 </li></ol> MDFチップの塩橋ネットワークの2セットアップ<ol><li>使用前に、121で15分間の保持時間を設定することにより、 図3Bに示されるすべてのプラスチックチューブ、指締めナット、およびマイクロ遠心チューブを滅菌 オートクレーブ中のCを°。 </li><li>媒体入口を経由してMDFチップアセンブリにフッ素樹脂チューブ（ 図3B-i）を接続します出口アダプタは、 図1Aに示します。 </li><li>媒体入口のルアーテーパーを接続し、3ウェイコックに図3B-iの中にプラスチックチューブ出口。 </li><li> 3 mlシリンジを用いてCO 2 -equilibratedリン酸緩衝生理食塩水（PBS）を2.5mlを取り、入口プラスチック管の3方コックに注射器を接続します。出口プラスチック管の3方活栓に、空の3 mlシリンジを接続します。 2つのシリンジは、このようにMDFチップを介して接続されています。 注：37にPBSをインキュベート C 5％-CO 2細胞培養インキュベータO / N°CO 2 を得るために PBSを-equilibrated。 </li><li>白色固体指締めナット（ 図3B-III）で、レイヤ1 PMMAシート上に青と緑のアダプタ（ 図1A）の開口部をシール。 </li><li> CO 2で塩橋チャネルおよび培養チャンバーを記入し、セットアップ手順2.4で説明したシリンジにPBSを-equilibrated。避ける気泡の形成。 </li><li>次に、CO 2を入れはO / Nインキュベーション37℃5％-CO 2細胞培養インキュベーター中でPBS含有MDFチップ-equilibrated。これは、両面テープの溶存空気が室内に気泡を形成することができます。 </li><li> 2つのシリンジを使用してチャネルの急速なPBSの流れによってチャネル内の気泡を洗い流します。必要に応じて前後にPBSをポンプ。 </li><li>媒体出口管に接続された3方活栓を介しチャンネルにPBSを排出します。 </li><li>新しい3-mlの注射器を使用して、CO 2の2.5ミリリットルを取るダルベッコ改変イーグル培地（DMEM）を（ステップ3.5を参照）-equilibratedと、新しいものと入口に接続注射器を交換してください。媒体とのチャネルを補充。 </li><li>塩橋ネットワークの調製。 <ol><li>時間的に（&gt; 70℃）、アガロース塩BRIに緑アダプタ（ 図1A）上（ 図3B-III）固体のナットを取り外して、3％のホット注入グリーンアダプタ（ 図1A）の開口部を通ってDGEチャンネル。 注：ホットアガロースの調製：PBS 50ml中のアガロースの粉末1.5グラムを溶解します。オートクレーブ中で121℃で20分の保持時間を設定することにより滅菌します。 </li><li>液体は塩橋のチャネルの長さの四分の三を埋める際にアガロースを注入停止します。 </li><li>アガロース注射後（ 図3B-III）固体ナットをねじ込むことによって緑アダプタ（ 図1A）に気孔をシール。 </li><li>半透明の筒状の指締めナット（ 図 3B）と青アダプタ（ 図1A）の固体ナットを交換してください。 </li><li>筒状のナット（ 図 3B-IV）にアガロース予備加熱し、3％をロードします。 </li><li>前筒状のナット（ 図3B-IV）中のAg / AgCl電極（ 図 3B-V）を埋め込 ​​みますアガロースの凝固。 </li></ol></li><li>塩橋ネットワークのセットアップが完了した後、培養室に一つずつ室CL1-5肺癌細胞を注射します。 </li></ol> 3.癌細胞の調製とElectrotactic実験用に設定<ol><li>肺癌細胞株CL1-5の正規培養。 <ol><li> 37で75T細胞培養フラスコ中の完全培地中で教授パンChyrヤン、17から得られた培養CL1-5肺癌細胞、 5％CO 2雰囲気下でC°。完全培地は、DMEM、10％ウシ胎児血清（FBS）で構成されています。サブカルチャー細胞ごとに3〜4日。走電性実験を行うために使用される細胞は、元のソースからの25未満の通路です。 </li></ol></li><li>指数関数的に増殖CL1-5細胞に0.25％トリプシン緩衝液2 mlを加え、37で2分間インキュベート 細胞detachmenため℃、5％-CO 2細胞培養インキュベータートン。 </li><li> 10％FBS DMEM 6 mlの脱離プロセスを終了し、室温で5分間300グラムで細胞を遠心します。その後、培地を廃棄し、PBS 5mlで細胞ペレットを懸濁します。 </li><li> PBS中の細胞数をカウントします。その後、室温で5分間、300gで1×10 6個の細胞と遠心分離機を取ります。 </li><li> PBSを捨てます。予め温めておいたCO 2 -equilibrated DMEMで細胞を懸濁し、1×10 6細胞/ mlであることが細胞密度を調整します。 注：37に完全培地をインキュベート C 5％-CO 2細胞培養インキュベータO / N°CO 2 を得た DMEMを-equilibrated。 </li></ol> 4. Electrotactic実験用に設定<ol><li> MDFチップの出口から、チップへの細胞の0.3ミリリットル、1×10 6 / mlで、注入します。 </li><li>細胞培養インキュベーター中で細胞を播種したMDFチップをインキュベート（37に設定 2-4時間、℃、5％CO 2）。 </li><li> MDFマイクロのセットアップ流体システム。 <ol><li>透明なインジウムスズ酸化物ガラス（ITO）、ヒーターの上にMDFマイクロ流体システムをインストールします。 MDFチップとITOガラスの間にクリップされたK型熱電対を持つMDFチップの温度を測定します。比例積分微分（PID）コントローラを用いてITOヒータを制御します。 37 MDFマイクロ流体システムのインキュベーション温度を設定します PIDコントローラとC°。 注：ITOヒーターの製造および細胞培養加熱システムのセットアップは、以前の報告に記載されている18,19。 </li><li>倒立顕微鏡にコンピュータ制御XYZ電動ステージ上の温度制御のMDFチップアセンブリをマウントします。電動ステージの使用は、ステップ5.1に記載されています。 4チャンネルシリンジポンプで注入口を介して培養室に完全培地をポンプ。 </li></ol></li><li> 20マイクロリットル/ hrの流量でMDFチップに完全培地をポンプ。外からの培養廃棄物（ 図3Aの 「廃棄物」として示される）マイクロ遠心チューブに回収することができます。 </li><li> MDFチップに37℃でさらに16〜18時間、細胞をインキュベートします。 </li><li>培地を交換するには、10分間、20μL/分の流速で各培養室に、それぞれ50、25、5、0μMキナーゼ阻害剤を含む培地ポンプ。 注：10.6 mgのキナーゼ阻害剤を、滅菌水3mlを添加することにより、Rho関連プロテインキナーゼ阻害剤Y27632の1​​0mMストック溶液を調製します。 10％FBS DMEM培地を用いて、それぞれ、50、25、及び5μMに10mMのストック溶液を希釈します。 </li><li> 37℃でさらに60分間、キナーゼ阻害剤で細胞をインキュベートします。 / hrで20μlに培地流量​​を設定します。同じ培養温度を使用し、後のステップで上記の流量。 </li><li>チップ上のAg / AgCl電極を介してMDFチップに直流電源を接続する電気配線を使用します。 </li><li>逐次電流計Iを接続n個の電気回路。 MDFチップの電流を監視するための電流計を使用してください。 </li><li> DC電源をオンにして、15〜19 Vに電源​​電圧を調整することにより、86.94μAの電流計電流を設定 注：オームの法則を考えると、私はバルク材料に流れる電流、 すなわち 、electrotacticチャンバ内の培地で、E = I /（σAのEFF）、σ（= 1.38Ω-1 M - 1）であります培地の導電率（0.07ミリ×高さ3mmの幅のために= 0.21 平方ミリメートル）effは electrotactic室の有効断面積であり、電流（I）は、300 MV / MMを生成するために必要MDFチップ内のEFは86.94μAです。 </li></ol> 5.細胞画像の取得と解析<ol><li> MATLABのGUIプログラムを使用して、電動ステージを制御します。 microscoに搭載されたMDFマイクロ流体システムでPEは、電動ステージを使用して観察領域にチップを移動します。 </li><li>デジタル一眼レフ（SLR）カメラを用いて記録する細胞画像は、顕微鏡に取り付けられました。 </li><li> 2時間15分の間隔で4倍の対物レンズを使用して顕微鏡画像を取ります。 </li><li>細胞遊走分析。 <ol><li> NIH ImageJの1.47 Vを実行します</li><li>セルの重心の最終位置に初期からの距離を分析します。 </li><li> 「ファイル」→「インポート」→「イメージシーケンス」に移動し、細胞遊走の分析のための9つの画像をインポートします。最初のイメージは、ゼロ時間の結果であり、最後の画像は、120分の結果です。 </li><li> →「 測定の設定」し、次の「 重心」のボックスをチェックし、「 分析」に進みます </li><li> 「 フリーハンド選択」アイコンをクリックします。 </li><li>最初の画像で選択されたセルのエッジを示しています。 </li><li> 「編集」に進みます→ </em&gt;「 選択」→「Manager への追加」 </li><li>第九の画像に移動し、最初のイメージに選択された同じ細胞のエッジを示しています。セル位置は、第八の画像の2番目のを使用して追跡することができます。 </li><li> →「Managerへの追加」「編集」→「選択」に進みます </li><li>繰り返し、90〜100個の細胞からデータを収集するために5.4.9に5.4.5を繰り返します。 </li><li>選択したセルの最初と最後の位置の結果を得るために→「分析」「測定」に進みます 。 注：移動速度は、時間当たりの平均細胞運動の長さとして定義されます。 directednessは、（陽極から陰極へ）DCEFのベクターおよびその最終位置への細胞の出発点からのベクトルの間の角度である余弦として定義されます。 directednessは陰極に向かって移動する細胞のための陽極に向かって移動する細胞のための-1、+ 1です。ランダムマイルのグループの場合細胞格子、directednessは0.2です</li></ol></li></ol>

<ol>
	<li>McCaig, C. D., Rajnicek, A. M., Song, B., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol Rev. 85, 943-978 (2005).</li><li>Djamgoz, M. B. A., Mycielska, M., Madeja, Z., Fraser, S. P., Korohoda, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+movement+of+rat+prostate+cancer+cells+in+direct-current+electric+field:+involvement+of+voltagegated+Na++channel+activity.">Directional movement of rat prostate cancer cells in direct-current electric field: involvement of voltagegated Na+ channel activity.</a> J Cell Sci. 114, 2697-2705 (2001).</li><li>Pu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+receptor+signaling+is+essential+for+electric-field-directed+migration+of+breast+cancer+cells.">EGF receptor signaling is essential for electric-field-directed migration of breast cancer cells.</a> J Cell Sci. 120, 3395-3403 (2007).</li><li>Huang, C. W., Cheng, J. Y., Yen, M. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+lung+cancer+cells+in+a+multiple-electric-field+chip.">Electrotaxis of lung cancer cells in a multiple-electric-field chip.</a> Biosens Bioelectron. 24, 3510-3516 (2009).</li><li>Huang, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+human+lung+cancer+cell+line+CL1-5+in+response+to+a+direct+current+electric+field.">Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.</a> PLoS One. 6, e25928 (2011).</li><li>Sun, Y. S., Peng, S. W., Lin, K. H., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+lung+cancer+cells+in+ordered+three-dimensional+scaffolds.">Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds.</a> Biomicrofluidics. 6, 14102-1410214 (2012).</li><li>Tsai, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+EGFR+and+RTK+signaling+in+the+electrotaxis+of+lung+adenocarcinoma+cells+under+direct-current+electric+field+stimulation.">Evaluation of EGFR and RTK signaling in the electrotaxis of lung adenocarcinoma cells under direct-current electric field stimulation.</a> PLoS One. 8, e73418 (2013).</li><li>Hou, H. S., Tsai, H. F., Chiu, H. T., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+chemical+and+electrical+stimulation+on+lung+cancer+cells+using+a+multichannel-dual-electric-field+chip.">Simultaneous chemical and electrical stimulation on lung cancer cells using a multichannel-dual-electric-field chip.</a> Biomicrofluidics. 8, (2014).</li><li>Faupel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropotential+evaluation+as+a+new+technique+for+diagnosing+breast+lesions.">Electropotential evaluation as a new technique for diagnosing breast lesions.</a> Eur J Radiol. 24, 33-38 (1997).</li><li>Szatkowski, M., Mycielska, M., Knowles, R., Kho, A. L., Djamgoz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+rat+prostate+gland+in+vitro:+identified+single-cell+and+transepithelial+(lumen)+potentials.">Electrophysiological recordings from the rat prostate gland in vitro: identified single-cell and transepithelial (lumen) potentials.</a> BJU Int. 86, 1068-1075 (2000).</li><li>McCaig, C. D., Song, B., Rajnicek, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+dimensions+in+cell+science.">Electrical dimensions in cell science.</a> J Cell Sci. 122, 4267-4276 (2009).</li><li>Das, T., Maiti, T. K., Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+on-chip:+shear+deformation+of+fibroblast+cells.">Traction force microscopy on-chip: shear deformation of fibroblast cells.</a> Lab Chip. 8, 1308-1318 (2008).</li><li>Lin, F., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+chemotaxis+in+a+simple+microfluidic+device.">T cell chemotaxis in a simple microfluidic device.</a> Lab Chip. 6, 1462-1469 (2006).</li><li>Li, J., Zhu, L., Zhang, M., Lin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+studying+cell+migration+in+single+or+co-existing+chemical+gradients+and+electric+fields.">Microfluidic device for studying cell migration in single or co-existing chemical gradients and electric fields.</a> Biomicrofluidics. 6, 24121-2412113 (2012).</li><li>Tsai, H. F., Peng, S. W., Wu, C. Y., Chang, H. F., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+oral+squamous+cell+carcinoma+cells+in+a+multiple-electric-field+chip+with+uniform+flow+field.">Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field.</a> Biomicrofluidics. 6, 34116 (2012).</li><li>Cheng, J. Y., Wei, C. W., Hsu, K. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+laser+micromachining+and+universal+surface+modification+of+PMMA+for+device+development.">Direct-write laser micromachining and universal surface modification of PMMA for device development.</a> Sensors and Actuators B: Chemical. 99, 186-196 (2004).</li><li>Chu, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+invasive+and+metastatic+subpopulations+from+a+human+lung+adenocarcinoma+cell+line.">Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line.</a> Am J Respir Cell Mol Biol. 17, 353-360 (1997).</li><li>Cheng, J. Y., Yen, M. H., Kuo, C. T., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transparent+cell-culture+microchamber+with+a+variably+controlled+concentration+gradient+generator+and+flow+field+rectifier.">A transparent cell-culture microchamber with a variably controlled concentration gradient generator and flow field rectifier.</a> Biomicrofluidics. 2, 24105 (2008).</li><li>Cheng, J. -. Y., Yen, M. -. H., Hsu, W. -. C., Jhang, J. -. H., Young, T. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITO+patterning+by+a+low+power+Q-switched+green+laser+and+its+use+in+the+fabrication+of+a+transparent+flow+meter.">ITO patterning by a low power Q-switched green laser and its use in the fabrication of a transparent flow meter.</a> Journal of Micromechanics and Microengineering. 17, 2316 (2007).</li><li>Pu, J., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+polarization+in+a+strong+electric+field.">Golgi polarization in a strong electric field.</a> J Cell Sci. 118, 1117-1128 (2005).</li><li>Zhao, M., Bai, H., Wang, E., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+directly+induces+pre-angiogenic+responses+in+vascular+endothelial+cells+by+signaling+through+VEGF+receptors.">Electrical stimulation directly induces pre-angiogenic responses in vascular endothelial cells by signaling through VEGF receptors.</a> J Cell Sci. 117, 397-405 (2004).</li><li>Yao, L., Shanley, L., McCaig, C., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J Cell Physiol. 216, 527-535 (2008).</li></ol>

The authors declare that they have no competing financial interests.

私たちは、トリッキーなことをMDFチップのレイヤー1の上にアクリルアダプタを付着させる工程を発見しました。スーパー接着剤のわずか1〜2μLの適用はしっかりとMDFチップ上にアダプターを接着するのに十分です。接着剤より多量のは、スーパー接着剤と接着するための失敗の不完全な重合をもたらしました。アクリルアダプタがしっかりとMDFチップ上に付着させた後は、マイクロ流体シス?...

直流電界（DCEF）の下で陽極または陰極に向かって移動し、接着細胞の挙動は、走電性と呼ばれます。細胞のelectrotactic挙動は、胚形成、神経再生、および創傷治癒において重要な役割を果たしている。そのようなラット前立腺癌細胞、2乳癌細胞、3および肺腺癌細胞を4-8として1腫瘍細胞を適用DCEF下electrotactic動きを示しています。生理学的EFは、腺組織において測定された。9,10走電性はまた、腺に関連する腫瘍細胞において報告されている。2,3まとめると、癌細胞の走電性は、転移因子であると考えられている。 図11は、電気的なガイダンスを制御しますDCEF下の癌細胞は、癌の将来の治療のための潜在的なアプローチであり得ます。しかし、今日、走電性の詳細な分子メカニズムは依然として議論の余地があります。したがって、INFの調査癌細胞遊走に対する電気刺激のluenceは、癌治療のための戦略の開発を容易にすることができます。 最近、バイオマイクロ流体デバイスは 、インビトロで、12化学勾配、13および電気刺激4にせん断力を流す細胞応答を研究するために作製されています。 （また、アクリル系として知られているPMMA）、ポリジメチルシロキサン（PDMS）またはポリメチルメタクリレートを使用して、バイオマイクロ流体デバイスの製造に成功し、そのような実験の失敗率が低下しています。さらに、生物学的対象を調査するためのプロトタイプとしてアクリル系のマイクロ流体デバイスを使用して、PDMSチップを使用するよりも簡単です。アクリルベースの​​デバイスで様々な機能が走電性研究のために開発されてきました。しかし、以前の設計のいずれも、同時に走電性研究のための細胞の種々の化学的条件や電界の影響をテストすることができません。したがって、我々は、マイクロ流体デバイスを開発したムーデュアル電界（MDF）チップ含有4つの独立した培養チャネルと1つのチップで8つの異なる実験条件をltichannel。 最初の侯らによって報告されたアクリル系MDFチップ、。、8は電気刺激し、いくつかの化学的に分離されたチャネルを統合しています。これらの化学的に単離されたチャネルは、1つの実験において培養する細胞の種類を使用することができます。チャンネル内DCEFは、電力供給によって生成されます。二つの独立した電場の印加電界強度（EFS）を有するものと0 EFSとは別の、それぞれの化学的に単離されたチャネルで行われます。このように、チップは、より良い制御の共存EFおよび化学的刺激を提供します。また、MDFチップ内部の化学拡散の数値シミュレーションの結果は、交差汚染は、24時間の実験期間の後にチャネル間で発生していないことを示している。8 デビと比較するとLiらによって報告されCE。、14 MDFチップを電気的に刺激された細胞のさらなる生化学的分析を可能にするより大きな培養面積を提供します。また、MDFチップの大きな観察領域に、より多くの細胞が、試験で観察することができるので、電気的に刺激された細胞の移動速度やdirectednessの分析はより正確です。 Huangらにより報告された以前の研究の単一チャネルチップ設計4、ツァイら 15細胞または化学物質の一種類のみを試験することを可能にします。ただし、MDFチップは走電性に様々な化学物質の影響、ならびに細胞の異なるタイプの電気刺激の効果を調査するために使用することができます。つまり、MDFチップは、化学線量依存性を効率的に研究することができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >12800-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Basic Life</td>
			<td >BL2651</td>
			<td></td>
		</tr>
		<tr >
			<td >Y-27632 (hydrochloride)</td>
			<td >Cayman Chemical Co</td>
			<td >10005583</td>
			<td></td>
		</tr>
		<tr >
			<td >agarose</td>
			<td >LONZO, USA</td>
			<td >SeaKem LE AGAROSE</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe</td>
			<td>Terumo</td>
			<td>3 ml with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td>Nipro</td>
			<td >with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >PMMA (acrylic)</td>
			<td >HiShiRon Industries CO., Ltd, Taiwan</td>
			<td ></td>
			<td >thickness 1mm, 2mm</td>
		</tr>
		<tr >
			<td >acrylic adaptor</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td >1/4-28 port, 10x10x6 mm</td>
			<td>customized</td>
		</tr>
		<tr >
			<td >nut</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope cover glass</td>
			<td >Deckgl&#228;ser, Germany</td>
			<td >24x60 mm</td>
			<td></td>
		</tr>
		<tr >
			<td >double-sided tape</td>
			<td >3M</td>
			<td >PET 8018</td>
			<td></td>
		</tr>
		<tr >
			<td >super glue</td>
			<td >3M</td>
			<td >Scotch Liquid Plus Super Glue</td>
			<td></td>
		</tr>
		<tr >
			<td >Teflon tube</td>
			<td >HENG YI ENTERPRISE CO., LTD., Taiwan</td>
			<td >UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING</td>
			<td>outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scienti&#64257;c</td>
		</tr>
		<tr >
			<td >TFD4 detergent</td>
			<td >Franklab, France</td>
			<td >TFD4</td>
			<td></td>
		</tr>
		<tr >
			<td >ultrasonic steri cleaner</td>
			<td >LEO ULTRASONIC CO., LTD., Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo bonder</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 laser scriber</td>
			<td >LTT group, Taiwan</td>
			<td >ISL-II</td>
			<td></td>
		</tr>
		<tr >
			<td >indium tin oxide glass (ITO glass)</td>
			<td >AimCore Technology Co., Ltd</td>
			<td >TN/STN, &#8806;10&#937;</td>
			<td></td>
		</tr>
		<tr >
			<td >proportional-integral-derivative (PID) controller</td>
			<td>JETEC Electronics Co., Japen</td>
			<td >TTM-J40-R-AB,</td>
			<td></td>
		</tr>
		<tr >
			<td >K-type thermocouple</td>
			<td >TECPEL</td>
			<td >TPK-02A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-channel syringe pump</td>
			<td >KdScientific, USA</td>
			<td >250P</td>
			<td></td>
		</tr>
		<tr >
			<td >DC power supply</td>
			<td >GWInstek, Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >X-Y-Z motor stage</td>
			<td >TanLian, E-O Co. Ltd., Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >inverted microscope</td>
			<td >Olympus, Japan</td>
			<td >CKX41</td>
			<td></td>
		</tr>
		<tr >
			<td >digital SLR camera</td>
			<td >Canon, Japan</td>
			<td >60D</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

直流電界（DCEF）の下方向の細胞遊走の挙動は走電性と呼ばれます。胚発生の間の細胞移動、細胞分化、および創傷治癒を誘導する生理的DCEFの重要な役割が多くの研究で実証されています。走電性アッセイにマイクロ流体チップを適用することによって、調査プロセスが短縮され、実験誤差が最小化されます。近年では、マイクロ流体デバイスは、ポリマー物質（例えば、ポリメチルメタクリレート、PMMA又はアクリル）またはポリジメチルシロキサン（PDMS）が広く、電気刺激に対する細胞の応答を研究に使用されてきたから成ります。しかし、PDMSデバイスを製造するために必要な多数の工程とは異なり、uidicチップFLアクリルミクロの簡単かつ迅速な構築は、デバイスの試作と生産の両方に適しています。しかし、報告されたデバイスのいずれも、同時化学とDCEの効率的な研究を促進していません細胞上のFの影響。本稿では、私たちのデザインと肺癌細胞の化学的および電気刺激の同時効果を調査するアクリル系マルチチャンネルデュアル電界（MDF）チップの製造を説明します。 MDFチップは、単一のテストでは、電気的/化学的刺激の8の組み合わせを提供します。チップを大幅に必要な実験時間を短縮するだけでなく、走電性の研究の精度を向上させるだけではなく。

MDF装置の製造および組み立て アクリル系MDFチップの模式図は 、図1Aに示されています。四アクリルシート、1つのカバーガラス、13アクリルアダプタ、両面テープ片が完了MDFチップ（ 図2D）の組み立てに使用されました。 MDF装置で唯一4つの独立した文化チャンネルがあります。しか?...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

マルチチャンネルデュアル電界マイクロ流体チップを用いて、肺癌細胞の走電性の研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

Research

JoVE Journal

Bioengineering

8.6K ビュー.  Academia Sinica. Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

ビデオ: Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Fluorescence detection methods for microfluidic droplet platforms

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

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

マイクロ流体液滴のプラットフォームのための蛍光検出法

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

生物工学

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

のオンデマンド定量分析のためのマイクロ流体ベースの走電性<em&gt;線虫（Caenorhabditis elegans）</em&gt; &#39;ロコモーション

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

マイクロ流体チップの作製とインフルエンザを検出する方法

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

単一細胞の多彩な化学分析マイクロ流体チップ

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic Chip for ICPMS Sample Introduction

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

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

ICPMSサンプル導入のためのマイクロ流体チップ

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

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多臓器チップ - 長期マルチ組織の共培養のためのマイクロ流体プラットフォーム

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

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 ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

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

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

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

マイクロ流体チップを使用した決定論的方法で単一細胞ベースの共培養を確立する

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...