著者は、財政支援のために彼らの初期の研究者賞プログラムを通じて自然科学とカナダの工学研究評議会、カナダリサーチチェアプログラム、保健研究のカナダの協会、および研究とイノベーションのオンタリオ州省に感謝したいと思います。

1。マスター金型製作のためのフォトリソグラフィー<ol><li> 30秒後、30秒、メタノール、アセトン、シリコンウェハー3インチ浴びる。 5分間のdH 2 0水で洗い流してください。 </li><li> N2ブローガンでウェハの表面を乾燥させる。 2分間140℃のホットプレート上のウェハを加熱。 </li><li>プラズマは、シリコンウェハ（1分間、50 W）の表面を酸化させる。 </li><li> 3ミリリットルSU-8フォトレジスト100（; 1,750 rpmで40秒）をスピンコートウェハの表面。 </li><li> 65℃のホットプレート上でプリベークコーティングウェーハ℃で10分間、その後95℃で2分間にわたって温度を上昇。さらに1時間のために、この設定を維持します。 </li><li>希望するチャネル設計を含むフォトマスクの位置を合わせます。 UV光の550から600 MJ / cm 2と （350-400 nm）のに抵抗を公開。フォトマスクは、AutoCADで設計され、高解像度の印刷で透明で印刷することができる。 </li><li> Tをランピングポストベーク65℃のホットプレート上のウエハ℃で10分間で1分、95℃でC、以前のように彼は温度。 </li><li> 10〜15分間SU-8現像液にウェハを浸し。イソプロパノールでリンスすることにより、開発の完了を確認してください。白色の沈殿物が表示された場合は、開発を継続。マスターモールドは、 図2Aに示されている。 </li></ol> 2。マイクロチャネル作製用ソフトリソグラフィ<ol><li> 3.5ミリリットルPDMS硬化剤と35ミリリットルポリジメチルシロキサン（PDMS）エラストマーベースを混ぜる。 </li><li>アルミホイルで裏打ちペトリ皿に製造されたマスターモールド（パターンが上向き）とブランクシリコンウエハーを置きます。 </li><li>マスター型皿と第二皿に15ミリリットルに20ミリリットルPDMSプレポリマーを注ぐ。優しく使い捨て木製のアプリケーターでそれらを押すことによってウェハの下にエアポケットを排除します。 </li><li>両方の料理をカバーし、治すための日に取っておく。あるいは2について、より速く硬化のために、真空脱気装置を用いたPDMSから気泡を除去した後、80℃のホットプレート上に皿を残す°CHR。 </li><li>ウェーハから箔とPDMS皮を取り除きます。 </li><li>チャネルの両端に流体アクセスポートをパンチするためにハリスユニコア（2.5 mm）を使用します。同様のサイズのストリップにチャンネルとブランクPDMSをカットします。 </li><li>プラズマ酸化剤にチャネル、ブランクPDMSストリップとスライドガラス（75×25ミリメートル2）をロード、おそらくクリーンルームに位置。 40 Wパワーで40秒間酸素プラズマにさらす。 </li><li>ブランクストリップの反対側へのチャネルピースとスライドガラスを貼り付けます。結合を完了するには2時間のために取っておく。 </li><li> 120℃のホットプレート上にアセンブリを置き℃にPDMSプレポリマーを使用してパンチの貯水池に、それぞれの長い少なくとも6インチ（内径1/32 &quot;、外径3/32&quot;）プラスチックチューブを取り付けます。一方または両方のチューブに貼り、流体プラスチックコネクタシリンジ添付ファイルを許可するか、市販の金具を使用する。 </li><li> PDMSは、治すためにチューブを固定できます。 EACに22ゲージの絶縁銅線の3 &quot;の長さを挿入hの入口管とチャネルの間に貯水池と、PDMSプレポリマーで固定します。完成品は、 図2Bに示されている。 </li></ol> 3。走電性実験<ol><li>モニタ（ 図1）に接続され搭載されたカメラと顕微鏡のステージ（望ましくはXY-可動）にマイクロチャネルを置きます。 </li><li>のマイクロ電極に電源やアンプの出力線を接続。 DC信号が所望される場合にのみ、単純なDC電源は十分であるが、関数発生器に接続された増幅器も同様にパルスDC及びAC信号の印加を可能にする。 </li><li>使い捨て注射器にマイクロチャネルの出力管を取り付けます。水没M9生理的緩衝液中入口管の口をゆっくりと注射器（手動またはシリンジポンプを用いて）内の負圧を適用してチャネルに液体を熱望する。入口および出口管はM9が充填されている両方の場合には、注射器fを切断するロムチューブ。静水圧駆動流を防止するために同じ高さの両方の管を水平。 </li><li>チャネルに直流電圧を​​印加し、その抵抗性を確保（R = V / I）は0.6MΩ（長さ50mm、幅の広い0.3ミリメートルとマイクロチャネルの深〜0.1ミリメートルの場合）程度である。 </li><li>チャンネルの整合性に満足している場合、チャネルに希釈懸濁液からワームをロードするには、上記の手順に従ってください。 </li><li>シリンジを外し、水圧管の相対的な高さを調整することにより、流れを操作します。チャネルの中心にワームを配置し、同じ高さで、両方のチューブが平らにするために、このメソッドを使用します。 </li><li> L3段動物のための4-12 V / cmのL4sための4-10 V / cmであり、若者のために2-4 V / cmで：適切な電圧の電源を設定します。電気信号をアクティブにして、フィールドに順応するためにワームの事前暴露の1分を可能にします。ワームは、陰極に向かって移動を開始する必要があります。分が経過すると、録音を開始するには、カメラを使用しています。 </li><li> ACおよびパルスに対する7,8を必要に応じて、dはDC実験では、最大応答電界が信号の前記周波数およびデューティサイクルから採用することができるが変調することができる。 </li><li>実験が終了すると、チャネルからすべての液体（やワーム）を削除、のdH 2 0でそれを洗い流し、そして℃の乾燥する125℃のホットプレート上のデバイスにしておきます。 </li><li>手動NIH ImageJの（使用して録画したビデオからの運動器官のデータを抽出<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ）またはカスタムMATLABベースのワームトラッキングソフトウェアを。 </li></ol>

<ol>
	<li>Kuwahara, T., Koyama, A., 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=Familial+Parkinson+mutant+&#945;-synuclein+causes+dopamine+neuron+dysfunction+in+transgenic+Caenorhabditis+elegans.">Familial Parkinson mutant &#945;-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.</a> J. Biol. Chem. 281 (1), 334-340 (2006).</li><li>Kuwahara, T., Koyama, A., 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=A+systematic+RNAi+screen+reveals+involvement+of+endocytic+pathway+in+neuronal+dysfunction+in+a-synuclein+transgenic.">A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in a-synuclein transgenic.</a> 17 (19), 2997-3009 (2008).</li><li>Su, L. J., Auluck, P. K., 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=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Dis. Model Mech. 3 (3-4), 194-208 (2010).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+sorting+of+Caenorhabditis+elegans.">Electrical sorting of Caenorhabditis elegans.</a> Lab Chip. 12 (10), 1831-1840 (2012).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pulse+direct+current+signals+on+electrotactic+movement+of+nematodes+Caenorhabditis+elegans+and+Caenorhabditis+briggsae.">Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae.</a> Biomicrofluidics. 5 (4), 044116 (2011).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Caenorhabditis+elegans+in+alternating+electric+field+and+its+application+to+their+localization+and+control.">Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control.</a> Appl. Phys. Lett. 96 (15), 153702 (2010).</li><li>van Ham, T. J., Thijssen, K. L., Breitling, R., Hofstra, R. M., Plasterk, R. H., Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+model+identifies+genetic+modifiers+of+alpha-synuclein+inclusion+formation+during+aging.">C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.</a> PLoS Genet. 4, e1000027 (2008).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Gabel, C. V., Gabel, H., Pavlichin, D., Kao, A., Clark, D. A., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+mediate+electrosensory+behavior+in+Caenorhabditis+elegans.">Neural circuits mediate electrosensory behavior in Caenorhabditis elegans.</a> J. Neurosci. 27 (28), 7586-7596 (2007).</li><li>Chuang, H. -. S., Raizen, D. M., Lamb, A., Dabbish, N., Bau, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoresis+of+Caenorhabditis+elegans.">Dielectrophoresis of Caenorhabditis elegans.</a> Lab Chip. 11 (4), 599-604 (2011).</li><li>Cronin, C. J., Mendel, J. E., Mukhtar, S., Kim, Y. -. M., Stirbl, R. C., Bruck, J., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+measuring+parameters+of+nematode+sinusoidal+movement.">An automated system for measuring parameters of nematode sinusoidal movement.</a> BMC Genet. 6, 5 (2005).</li><li>Mani&#232;re, X., Lebois, F., Matic, I., Ladoux, B., Meglio, J. -. M. D. i., Hersen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Running+worms:+C.+elegans+self-sorting+by+electrotaxis.">Running worms: C. elegans self-sorting by electrotaxis.</a> PLoS One. 6 (2), e16637 (2011).</li></ol>

マイクロ流体走電性アッセイ技術は、アメリカとカナダ、米国で特許を申請しました。

最初荘や同僚11,12の誘電泳動操作作業にガベルと同僚や建物によって記述行動現象を利用して、当社のマイクロ流体ベースの走電性アッセイは、動きをとして使用してワームに神経活動を調べるため、簡単に堅牢かつ感度の高い方法を提供しています出力。運動パラメータの解析は異なる遺伝子型間の定量的な比較を可能にします。マイクロ流路の作製と電界印加の精度が一緒に制御...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CALEDON Labs</td>
			<td>1200-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CALEDON Labs</td>
			<td>6700-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>CALEDON Labs</td>
			<td>8600-1-40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Microchem Corp.</td>
			<td>Y131273</td>
			<td>SU-8 100</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem Corp.</td>
			<td>Y020100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92x16 mm Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>Contains elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Tektronix Inc.</td>
			<td>&nbsp;</td>
			<td>Model AFG3022B</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Trek Inc.</td>
			<td>&nbsp;</td>
			<td>Model 2210-CE</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4506</td>
			<td>Model 11 ELITE</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>11675916Q</td>
			<td>Model HP131725Q</td>
		</tr>
	</tbody>
</table>

microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

線虫線虫（Caenorhabditis elegans）があるため、疾患関連遺伝子および経路と同様に栽培の容易さのその保全の生物医学研究のための多目的なモデル生物である。いくつかのC.線虫病モデルは、例えばドーパミン作動性（DA）ニューロン1の変性を伴うパーキンソン病（PD）などの神経変性疾患を含む、報告されている。導入遺伝子と神経毒性化学物質の両方が神経保護遺伝子や化合物の2,3のための神経変性と画面の基礎の調査を可能にし、ワームでDA神経変性とそれに伴う動きの欠陥を誘発するために使用されている。 C.のような下等真核生物の画面線虫は、神経信号伝達に影響を与える化合物及び遺伝子を同定するための効率的かつ経済的な手段を提供する。従来の画面は、通常手動で実行して目視で採点され、その結果、彼らは時間両論ですumingとヒューマンエラーを起こしやすい。さらに、細胞レベルの分析で最もフォーカスが移動を無視して、これは、運動障害のために特に重要なパラメータである。 我々はC.を制御し、定量化した新規マイクロ流体スクリーニングシステム（ 図1）を開発しましたマイクロチャネル内部電界刺激を用いエレガンス &#39;ロコモーション。我々は、直流（DC）は、フィールドが確実にカソードに向かって移動（ &quot;走電性&quot;）4オンデマンド誘導することが示されている。フィールドの極性を逆にすると、ワームがすぐに同様にその方向を反​​転させる。また、ドーパミン作動性や他の感覚ニューロンの欠陥は、スイミングレスポンス5を変更することが示されている。したがって、ニューロンのシグナル伝達における異常が読み出しとして歩行を用いて決定することができる。運動応答は、正確にそのような遊泳速度、本体の曲げ周波数および反転時間などのパラメータの範囲を使用して定量することができる。 4に比べて速く移動。これらの知見は、私たちが受動的に年齢や表現型6でワームをソートする新しいマイクロ流体デバイスを設計するために導いた。 また、パルスDCにワームの応答をテストし、電流（AC）電界を交互にしています。 Cの両方でさまざまなデューティ·サイクルのパルスDCフィールドは、効果的に生成された走電性エレガンスとそのいとこでbriggsae 7。別の実験で、1ヘルツから3キロヘルツまでの周波数を持つ対称ACフィールドはチャンネル8内ワームを固定化した。 マイクロ流体環境における電界の実装では、走電性アッセイの迅速かつ自動実行を可能にする。このアプローチは、因子のハイスループット遺伝的および化学的な画面を容易にすることを約束神経機能および生存に影響を与える。

ワームトラッキングソフトウェアから野生型若い成人線虫の走電性とその位置と速度出力の代表的な映像は補足ビデオ1及び図3に示します。運動解析ソフトウェア自体は、フィールド極性の方向と極性反転の時間を認識していない、むしろ、この情報は、ソースビデオから取得する必要があります。これは、ビデオ、オーディオやビジュアルキューを使用したり、実?...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

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

にオンデマンド歩行を誘導する半自動微小電気流体方式<em&gt;線虫（Caenorhabditis elegans）</em&gt;記載されている。この方法は、マイクロ流体チャネル内部の穏やかな電場（ &quot;走電性&quot;）に応答するワームの神経生理学的現象に基づいている。マイクロ流体走電性は、ニューロンの健康に影響を与える要因のための画面に迅速、高感度、低コスト、かつスケーラブルな手法として機能します。

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-based-electrotaxis-for-on-demand-quantitative-analysis

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

9.7K ビュー.  McMaster University. にオンデマンド歩行を誘導する半自動微小電気流体方式線虫（Caenorhabditis elegans）記載されている。この方法は、マイクロ流体チャネル内部の穏やかな電場（ "走電性"）に応答するワームの神経生理学的現象に基づいている。マイクロ流体走電性は、ニューロンの健康に影響を与える要因のための画面に迅速、高感度、低コスト、かつス...

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

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

マイクロパターン細胞を用いた薬物の効果の改善可視化と定量分析

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

生物工学

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

単一粒子のためのマイクロ流体ベースの流体力学的トラップ

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

自由レーザー回折を用いた微生物水泳の定量的ロコモーションスタディ

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

SENP1プロテアーゼキネティクス決定のための定量的なFRET（フェルスター共鳴エネルギー移動）の分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

アデニリルシクラーゼの薬物誘発性感作：アッセイは、小分子およびsiRNAスクリーニング用途のために合理化·小型化

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

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 ...

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

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

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 ...

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 ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

機能再生大腿骨につながるラットモデルにおける仮骨延長法のための効率的かつ再現可能なプロトコル

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

カエノラブディティス ・ エレガンスにおける縦断的イメージングのためのマイクロ流体プラットフォーム

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...