私たちは、MABのデバイスのワイヤボンディングのための資金支援（助成番号103498と103692）、パーデュー大学のBirck NantechnologyセンターガーレロックウッドのためにNASA宇宙生物学科学技術楽器開発（ASTID）プログラムに感謝したい、とヨンジュンヒョンParkのでしょうフローセル室のCAD図面。

1。ポリの調製（3,4 -エチレンジオキシチオフェン）：ポリ（ナトリウム4 -スチレン）（PEDOT：PSS）H +とCO 3 2用電解ソリューション-イオン<ol><li>完全に分散するまで（約10秒）10ミリリットル脱イオン（DI）水と渦に- 70mgのポリ（ナトリウム4 -スチレン）（のNa + PSS）を追加します。 </li><li>溶液が完全に混合されるまで、1.1のソリューション、ボルテックスに10.7μlの3,4 - ethlyenedioxythiophene（EDOT）を追加します。 </li></ol> 2。硫酸カルシウム（PEDOT：のCaSO 4）のCa 2用電解液+イオンポリ（3,4 -エチレンジオキシチオフェン）の調製<ol><li> 10ミリリットルのDI水と渦に136 mgのカルシウム硫酸（のCaSO 4）を追加し、ソリューションは、完全に分散させると乳白色表示されません。 </li><li>完全に混合されるまで、2.1、ボルテックスで液に10.7μlのEDOTを追加します。 </li></ol> 3。 PEDOTベースの電解導電性ポリマー<ol><li>バイオ分析システムズ（BASI）C3セルスタンド（ 図3）およびECイプシロンポテンショスタット/ガルバノスタット電解ための電気化学セルを形成するために使用される。溶存酸素を除去するために20分間電気化学セル及び窒素バブルでPSS電解液：EDOTを置きます。 </li><li>現在電気化学セルの対向電極の位置に白金ガーゼをクリップ。次いで、白金ガーゼに対向する作用電極との電気化学セルの作用電極の位置にMABをクリップ。 PSS電解液：のみ円形の電極をPEDOTに沈めれるようにMABの深さを調整します。平方電気接触パッドを使用してソリューションの接触を避ける。 </li><li> BASI飽和銀/塩化銀（Ag / AgCl電極）電気化学セルの参照電極位置に電極を配置します。参照電極は、作業とcounteの間に含まれていないことを確認してくださいrは電極。 </li><li> PEDOTの場合：PSS堆積：20分間バブル電気化学セルを、と0Vから単一のサイクリックボルタモグラムを実行するためにECイプシロンポテンショスタット/ガルバノスタットを使用 - ±100μAスケールで20 mVの/秒のスキャンレートで1.1V。 </li><li>ためのPEDOT：のCaSO 4堆積：バブル20分間電気化学セル、および30分間nAの814でクロノを実行するためにECイプシロンポテンショスタット/ガルバノスタットを使用しています。 </li></ol> 4。 K 3サンタフェのPEDOT系ポリマー複合体のサイクリックボルタンメトリー（CN）6 <ol><li> 3.1から3.3まで、上記の手順を実行します。 </li><li> ±10μAスケールでMV /秒（25、50、75、100、L25、150、175、200）のスキャン速度を変えて-653 mVのから853 mVまでシングルサイクリックボルタモグラムを実行するためにECイプシロンポテンショスタット/ガルバノスタットを使用してください。 </li></ol> 5。表面機能化プロトコル<ol><li>ステップ3のように、興味のあるイオンの特定預金導電性ポリマー共役。 </li><li>ステップ6のように、イオン選択膜を適用します。 </li></ol> 6。イオン選択性膜の応用<ol><li>真空スピナーチャック上センターMAB。 </li><li> MABと実行の中心への預金100μlの膜。 </li><li>上下に5秒のランプで30秒間1,500 rpmでスピンコーターをスピンコートイオン選択性膜。 </li><li> 30分間スピンコートMABに掃除機をかける、20分間70℃のオーブンでチップを焼く。 </li></ol> 7。 pHと炭酸とPEDOT-PSS導電性高分子複合体（CO 3 2 - ）のキャリブレーションイオン選択性膜<ol><li>条件10μMの炭酸水素ナトリウム（NaHCO 3を ）と藻類のメディアで5 mMの塩化カリウム（塩化カリウム）でMAB一晩。 </li><li>マイクロ流体フローセルチップホルダーにMABを挿入します。 </li><li>初期pH値または濃度（CO 3 2用などのpH 4または10μM - ）で5ミリリットルの試験溶液を注入する。 BUBを削除フローセルチップホルダーからBLES。 </li><li>フローセル電気治具上にフローセルチップホルダーを置きます。 </li><li> ECイプシロンソフトウェアを開き、開回路電位（OP）モードに入ります。 300分、±1Vの電圧スケール、および10 kHzのカットオフ周波数までの時間を設定し、値ごとに2秒を記録します。 </li><li> MABは、キャリブレーション処理を続行する前に、（フラットラインを探してください）​​安定させます。 </li><li> MABが安定したら、試験溶液をフローセルをフラッシュし、校正されるべき次の濃度を注入液（pH5又は3 CO 2を 25μM - ）。全く泡がフローセルに入ることを許可されていないことを確認してください。 pHが6、7、8、9またはCO 3 2について、ステップ7.5お ​​よび7.6を繰り返す- 50の濃度、75、100、250、500、750、および1,000μM。 </li><li>最後の濃度が実行された後、窒素空気とMABとドライを削除します。 </li><li>次の使用まで、新鮮な空調ソリューションにMABバックを置きます。 </li></ol>塩化カルシウムでのCaSO 4導電性高分子共役<ol><li>条件0.1 MのCaCl 2および10μMのNaNO 3 7mlの中で一晩MAB。 </li><li> 7.10から7.2に似た手順に従ってください。ステップ8.3で、0.01のCaCl 2の初期濃度と炭酸テストソリューションを交換してください。 0.05、0.1、0.5、1および10mMの試験溶液濃度について、この手順を繰り返します。 </li></ol>

<ol>
	<li>Migdalski, J., Bas, B., Blaz, T., Golimowski, J., Lewenstam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Miniaturized+and+Integrated+Galvanic+Cell+for+the+Potentiometric+Measurement+of+Ions+in+Biological+Liquids.">A Miniaturized and Integrated Galvanic Cell for the Potentiometric Measurement of Ions in Biological Liquids.</a> J. Solid State Electrochem. 13, 149-155 (2009).</li><li>Buehler, M. G., Kounaves, S. P., Martin, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+Water-quality+Monitor+with+Ion-selective-electrodes.">Designing a Water-quality Monitor with Ion-selective-electrodes.</a> 1, 331-338 (2001).</li><li>Adamchuk, V. I., Lund, E. D., Sethuramasamyraja, B., Morgan, M. T., Doberman, A., Marx, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Soil+Chemical+Properties+on-the-go+using+Ion-selective-electrodes.">Direct Measurement of Soil Chemical Properties on-the-go using Ion-selective-electrodes.</a> Journal Computers and Electronics in Agriculture. 48 (3), 272-294 (2005).</li><li>Oel&#946;ner, W., Hermann, S., Kaden, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Sensors+and+Sensor+Module+for+Studying+Biological+Systems+in+Space+Vehicles.">Electrochemical Sensors and Sensor Module for Studying Biological Systems in Space Vehicles.</a> Aerospace Science and Technology. 1, 291-296 (1997).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+Polymer-based+Solid-state+Ion-selective+Electrodes.">Conducting Polymer-based Solid-state Ion-selective Electrodes.</a> Electroanalysis. 18 (1), 7-18 (2006).</li><li>Buck, 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> Ion Selective Electrodes in Analytical Chemistry. , (1980).</li><li>Nam, H., Cha, G. S., Yang, V. C., Ngo, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Biosensors and their Applications. , (2000).</li><li>Anatova-Ivanova, S., Mattinen, U., Radu, A., Bobacka, J., Lewenstem, A., Migdalski, J., Danielewski, M., Diamond, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Miniature+All-solid-state+Potentiometric+Sensing+System.">Development of Miniature All-solid-state Potentiometric Sensing System.</a> Sensors and Actuators B. 146, 199-205 (2010).</li><li>Michalska, A., Galuszkiewicz, A., Ogonowska, M., Ocypa, M., Maksymiuk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDOT+Films:+Multifunctional+Membranes+for+Electrochemical+Ion+sensing.">PEDOT Films: Multifunctional Membranes for Electrochemical Ion sensing.</a> J. Solid State Electrochem. 8, 381-389 (2004).</li><li>Bard, A. J., Faulkner, L. R., ed, 2. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electrochemical Methods: Fundamentals and Applications. , (2000).</li><li>Claussen, J. C., Artiles, M. S., McLamore, E. S., Mohanty, S., Shi, J., Rickus, J., Fisher, T. S., Porterfield, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Glutamate+Biosensing+with+Naanocube+and+Nanosphere+Augmented+Single-walled+Carbon+Nanotube+Networks:+A+Comparative+Study.">Electrochemical Glutamate Biosensing with Naanocube and Nanosphere Augmented Single-walled Carbon Nanotube Networks: A Comparative Study.</a> J. Mater. Chem. 21, 11224-11231 (2011).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+Stability+of+All-solid-state+Ion-selective+Electrodes+using+Conducting+Polymers+as+Ion-to-electron+Transducers.">Potential Stability of All-solid-state Ion-selective Electrodes using Conducting Polymers as Ion-to-electron Transducers.</a> Anal. Chem. 71, 4932-4937 (1999).</li><li>Lee, J. H., Yoon, I. J., Yoo, C. L., Pyun, H. J., Cha, G. S., Nam, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiometric+Evaluation+of+Solvent+Polymeric+Carbonate-selective+Membranes+based+on+Molecular+Tweezer-type+Neutral+Carriers.">Potentiometric Evaluation of Solvent Polymeric Carbonate-selective Membranes based on Molecular Tweezer-type Neutral Carriers.</a> Anal. Chem. 72, 4694-4699 (2000).</li><li>Song, F., Ha, J., Park, B., Kwak, T. H., Kim, I. T., Nam, H., Cha, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-solid-state+Carbonate+Selective+Electrode+based+on+a+Molecular+Tweezer-type+Neutral+Carrier+with+Solvent-soluble+Conducting+Polymer+Solid+Contact.">All-solid-state Carbonate Selective Electrode based on a Molecular Tweezer-type Neutral Carrier with Solvent-soluble Conducting Polymer Solid Contact.</a> Talanta. 57, 263-270 (2002).</li></ol>

我々は、開示することは何もありません。

MABバイオチップは、Pt電極、測定可能な電気信号に関心のイオン濃度を伝達したとの組合せにPEDOTベースCP共役伝達層の上にISMから構成さASSISEsで構成されています。安定した電極電位はCP層とISM層の両方によって定義される。両方の層はまた、MABと測定された電気信号の品質（ノイズ、ドリフト）の作業寿命を決定する。 PEDOTは、そのイオンと電子特性（その酸化形態で?...

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the items</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		 </tr>
		 <tr>
			<td>3,4-Ethylenedioxythiophene</td>
			<td>Sigma-Aldrich</td>
			<td>483028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly(sodium 4-styrenesulfonate)</td>
			<td>Sigma-Aldrich</td>
			<td>243051</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EC epsilon galvanostat/potentiostat</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>e2P</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Saturated Ag/AgCl reference electrode</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>MF-2052</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pt gauze</td>
			<td>Alfa Aesar</td>
			<td>10283</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P-8131</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium nitrate</td>
			<td>J.T. Baker</td>
			<td>3190-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate</td>
			<td>Mallinckrodt/ Macron</td>
			<td>7412-12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S-7127</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride</td>
			<td>J.T. Baker</td>
			<td>1311-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>237132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C3 cell stand</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>EF-1085</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flow-cell chip holder</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames</td>
		 </tr>
		 <tr>
			<td>Flow-cell electrical fixture</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Specific reagents and equipment.</td>
		 </tr>
		</tbody>
 </table>

multi-analyte biochip (mab) based on all-solid-state ion-selective electrodes (assise) for physiological research

環境、医学、農業、生物学、そして宇宙飛行研究のラボオンチップ（LOC）のアプリケーションは、複雑な生物学的メディア1-4に長期保管に耐えることができ、イオン選択性電極（ISE）が必要です。全固体型イオン選択性電極（ASSISE）は、前述の用途に特に魅力的である。バッチ処理を可能にし、容易に構築、低メンテナンス、および（の可能性）小型化：電極は、以下の有利な特性を持つ必要があります。微細加工ASSISE H +、Ca 2 +のを定量化するためのもの、と3 CO 2を-イオンが建設された。これは、貴金属電極層（ すなわち Pt）から、伝達層と、イオン選択性膜（ISM）層からなる。伝達層の機能は、測定可能な電気信号にイオン選択性膜の濃度依存性の化学ポテンシャルを伝達する。 T彼ASSISEの寿命は5-7導電層/膜界面の電位を維持することに依存することがわかった。 ASSISEワーキング寿命を延長することにより、界面層に安定した電位を維持するために、我々は（PEDOT）銀/塩化銀の代わりに7-9（銀/塩化銀）の導電性高分子（CP）、ポリ（3,4 -エチレンジオキシチオフェン）を利用変換器層として。我々は、多検体バイオチップ（MAB）（ 図1）と呼ばれるラボオンチップ形式でASSISEを構築した。 （測定範囲0.01ミリメートル- - 1 mM）を、およびCa 2 +（1 mMのに対数線形範囲0.01 mM）をテスト·ソリューションの校正は、MABはpH値（動作範囲のpHは4-9）、CO 3 2を監視することができることを実証した。 pHのためのMABは藻類培地のほぼ1ヶ月保存した後に近いネルンストスロープ応答を提供します。炭酸塩バイオチップは、従来のイオン選択性電極と同様の電位差プロファイルを示す。生理学ogical測定は、微細藻類クロレラ尋常性モデル系の生物学的活性を監視するために用いられた。 MABは、サイズ、汎用性に利点を伝え、そして検体センシング機能を多重化、地球上または空間内で、多くの閉じ込められた監視状況にそれが適用されること。 バイオチップの設計と実験方法バイオチップは、ディメンションで10×11ミリメートルで、作用電極（WES）と5のAg / AgCl参照電極（RES）として指定された9 ASSISEsを持っています。各ワーキング電極（WE）、直径240μmでかつ平等に、直径480ミクロンであるREを、から1.4ミリメートルに離間している。これらの電極は、0.5ミリメートル×0.5mmの寸法を有する電気接触パッドに接続されている。概略を図2に示されている。 サイクリックボルタンメトリー（CV）と定電流堆積方法はBioanalytic PEDOTを使用してフィルムをelectropolymerizeするために使用されるアル·システムズ（BASI）C3セルスタンド（ 図3）。 PEDOTフィルムの対イオンは、目的の分析物イオンに合わせて調整される。ポリ（スチレンスルホン酸）対イオン（PEDOT / PSS）とPEDOTは、H +及びCO 3 2のために利用される- 、硫酸（のCaSO 4として溶液に添加）と、ワンは、Ca 2 +のために利用されている。 PEDOT-コーティングの電気化学的特性は、WEは、レドックス活性溶液（フェリシアン化カリウムすなわち 2mMの（K 3のFe（CN）6））でCVを用いて分析されている。 CVプロファイルに基づいて、ランドレス·シェフチク分析は、有効表面積10を決定した。 1,500 rpmでスピンコーティングはMABワーキング電極（WES）で〜2μmの厚さのイオン選択膜（ISMS）をキャストするために使用されます。 MABは、藻類媒体150μlの体積を充填したマイクロ流体フローセルチャンバー中に含まれ、コンタクトパッドを電気的に（ 図 BASIシステムに接続されているURE 4）。 クロレラブルガリスの光合成活性は、周囲の明暗条件で監視されています。

<html> PEDOTのサイクリックボルタモグラム（CV）結果の例：PSSとそれに対応するカソードピーク電流（I P）対スキャン速度（V 1/2）は、それぞれ図5aおよび5bに示されている。 PEDOT：様々なスキャンレートおよびそのカソードピーク電流でのCaSO 4は表示されません。 PSSとPEDOT：ランドレス-シェフチク分析10、固体接触PEDOTの実...

Watch this Scientific Journal Video about Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research at JoVE.com

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

導電性高分子（CP）トランスデューサから構築全固体型イオン選択電極（ASSISEs）液体培地における機能生涯の数ヶ月を提供します。ここでは、ラボオンチップ形式でASSISEsの製作と校正プロセスについて説明します。 ASSISEは、複雑な生物学的培地で長期保管した後、近ネルンスト傾斜プロファイルを維持していることが実証されています。

生理学的研究のための全固体型イオン選択性電極（ASSISE）に基づき、多検体バイオチップ（MAB）

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

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Bioengineering

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

17.1K ビュー.  Purdue University. 導電性高分子（CP）トランスデューサから構築全固体型イオン選択電極（ASSISEs）液体培地における機能生涯の数ヶ月を提供します。ここでは、ラボオンチップ形式でASSISEsの製作と校正プロセスについて説明します。 ASSISEは、複雑な生物学的培地で長期保管した後、近ネルンスト傾斜プロファイルを維持していることが実証されています。

ビデオ: Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

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 ...

生物工学

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

回生研究ハーモニックナノ粒子

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

ラベルフリーDNAハイブリダイゼーション分析のためのマイクロ流体ベースの電気化学バイオチップ

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

ヒトアデノウイルス5型に基づく高容量アデノウイルスベクターのクローニングおよび大規模生産

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

バイオインスパイアードデザインのためのプロトコル：グランドサンプラーウニジョーズに基づいて、

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

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 ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

作製と内皮下電気抵抗を直接定量化する統合型電極を用いたオンチップ器官システムの検証

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

セルロースナノファイバーの一方向凍結乾燥法により作製した Microhoneycomb モノリス基づくゾル: メソッドと拡張機能

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

カーブレット変換ベースのツールを使用したフィブリラーコラーゲンの定量化

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

Microtubule Plus-End Dynamics Visualization in Huntington&#039;s Disease Model based on Human Primary Skin Fibroblasts

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

ヒト原発性皮膚線維芽細胞に基づくハンチントン病モデルにおける微小管プラスエンドダイナミクスの可視化

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...