この作品は、食料農業USDA国立研究所、国立競争補助金プログラム、国家研究イニシアティブプログラム71.1 2007年度によって部分的に資金を供給されているアレルギー感染病に対する認可番号2007-35503-18392、および国立衛生研究所、研究所など、R33AI94514-03。

1スピニングドープ準備<ol><li>調査されるべき生体高分子濃度の範囲を準備します（0.1％〜30％、w / v）の、これらの計算では、バイオポリマー粉末の含水率を考慮してください。各濃度について50mlの試験管にバイオポリマー（デンプン、プルラン）粉末の重量を量る。水性ジメチルスルホキシド（DMSO）溶液と撹拌棒を加える。 </li><li>マグネチックスターラーホットプレート上で一定に攪拌しながら沸騰したお湯にチューブを置きます。 </li><li>約1時間後、加熱をオフにし、分散液が室温に冷却させる。次に、分散液をレオロジーテストやエレクトロスピニングの準備ができています。 </li></ol> 2定常せん断レオロジー<ol><li>レオメーターをウォームアップし、20℃のステージ温度を設定します。プローブ（使用25mmのコーン）とステージ（プレート）の間のギャップを校正します。 </li><li>ステージの中央への生体高分子分散の荷重0.41ミリリットルとPOSITIを設定するためのプローブを下げる（25mmのコーンのための0.053ミリメートルギャップ）について。分散が均等にギャップ内に広がっていることを確認します。 </li><li>掃引モード：ログインし、初期速度：100秒-1、最終速度：0.1秒-1、十年ごとのポイント：10、対策前の遅延：5秒、小節時間：10秒、以下の実験パラメータでレオロジー試験を行う拍子や方向：2（反時​​計回りの両方）。 </li><li>試験をエレクトロスピニングするための適切な分散濃度を推定するレオロジーデータを分析します。 <ol><li>ポリマー濃度の関数としてせん断速度に対する見掛けのせん断粘度をプロットします。 0.1秒での見かけの粘度（低せん断速度のデータは信頼性が低い低濃度で、 例えば ）実際の、または外挿した値で、各フローカーブ、おおよそのゼロせん断粘度、η0、の場合- 1。 </li><li>比粘度を計算し、ηSP =（η0 - η 秒 ）ηsは溶媒の粘度である/ηsで、。 </li><li>濃度の関数として特定の粘度をプロットします。 semidilute交絡と絡み合った体制を識別します。 semidilute交絡政権は、小さな傾きで低濃度端から始まり、semidiluteもつれ政権は交絡政権、次のより大きな傾斜を有する。双方の体制に合わせるべき乗則回帰モデル。電力値は、両対数プロット上semidilute交絡と絡み合った体制での斜面（濃度依存性）である。 2近似直線の交点が絡み合い濃度はC eは 。 </li></ol></li></ol> 3エレクトロスピニングパラメータ変動<ol><li> 図1に示すように電界紡糸セットアップを組み立てます。シリンジポンプ上に、100％のDMSO中の15％（w / v）のデンプンまたはプルラン、 たとえば 、適切な組成の分散体と注射器をロードします。 POSIT（高電圧ワイヤークリップ針にIVE）。浴にアース線（マイナス）を浸漬することによって地面に純粋なエタノールを含む凝固槽を接続します。注射針と凝固浴との間の距離を調整するためにラボジャッキを使用してください。エレクトロスピニング後に繊維マットを収集する浴中に金属メッシュを浸す。 </li><li>次のパラメータに生体高分子をスピン範囲：0〜15 kVの0.1〜0.4ミリリットル/時間から速度、5〜10センチメートルスピニング距離、電圧を養う。 <ol><li> 5cmのスピニング距離から始めます。最初の送り速度（0.1ミリリットル/時）の場合は、0 V針の先端で押し出し、分散液の形状に注意を払い、滴下分散が加速した後、伸長した際に注意してから、ゆっくりと電圧が上昇します。 </li><li>小さなジェットは、溶液のelectrospinnabilityを示す、落下面から開始する電圧に注意してください。もしあれば、継続的なジェットが開始する電圧を記録します。 </li><li> 3 parameteのそれぞれのための完全な範囲を調べRSとノート成功紡糸条件。先端から連続ジェットがある場合にのみ繊維を収集します。 </li></ol></li><li>収集の数分後、純粋なエタノールで繊維マットをすすぐ。真空下で乾燥剤入りデシケーター中に繊維マットを置きます。 </li><li>完全な特性のために、各生体高分子濃度について繰り返します。 </li></ol><img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> エレクトロ湿式紡糸セットアップの図1概念図。バイオポリマー分散液をシリンジポンプから押し出される。高電圧DC電源は、鈍針を接地凝固浴に高電圧を提供する。針の先端からのポリマージェットはまっすぐな道を通って移動した後、急速なホイップ·パス（別名​​泡立て不安定）を開発しています。 4。形態学的特性評価<ol><li>乾燥した繊維マットの部分をカットし、カーボンテープを使用したSEMスタブの上に固定化する。 </li><li> SEM装置内に試料スタブをロードし、分析のための画像を取得する。 </li></ol>

<ol>
	<li>Greiner, A., Wendorff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+self-assembled+nanofibers+by+electrospinning.">Functional self-assembled nanofibers by electrospinning.</a> Self-aseembled nanomaterials. 1, 107-171 (2008).</li><li>Ramakrishna, S., Fujihara, K., Teo, W. E., Lim, T. C., Ma, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Electrospinning and Nanofibers. , (2005).</li><li>Klossner, R. R., Queen, H. A., Coughlin, A. J., Krause, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Chitosan&#8217;s+Rheological+Properties+and+Its+Ability+to+Electrospin.">Correlation of Chitosan&#8217;s Rheological Properties and Its Ability to Electrospin.</a> Biomacromolecules. 9 (10), 2947-2953 (2008).</li><li>McKee, M. G., Wilkes, G. L., Colby, R. H., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+of+Solution+Rheology+with+Electrospun+Fiber+Formation+of+Linear+and+Branched+Polyesters.">Correlations of Solution Rheology with Electrospun Fiber Formation of Linear and Branched Polyesters.</a> Macromolecules. 37 (5), 1760-1767 (2004).</li><li>McKee, M. G., Hunley, M. T., Layman, J. M., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+rheological+behavior+and+electrospinning+of+cationic+polyelectrolytes.">Solution rheological behavior and electrospinning of cationic polyelectrolytes.</a> Macromolecules. 39 (2), 575-583 (2006).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+pure+starch+fibers+by+electrospinning.">Fabrication of pure starch fibers by electrospinning.</a> Food Hydrocolloids. 36, 20-25 (2014).</li><li>Singh, R. S., Saini, G. K., Kennedy, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pullulan:+microbial+sources,+production+and+applications.">Pullulan: microbial sources, production and applications.</a> Carbohydrate Polymers. 73 (4), 515-531 (2008).</li><li>Leathers, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+and+applications+of+pullulan.">Biotechnological production and applications of pullulan.</a> Applied Microbiology and Biotechnology. 62 (5), 468-473 (2003).</li><li>Karim, M. R., Lee, H. 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=Preparation+and+characterization+of+electrospun+pullulan/montmorillonite+nanofiber+mats+in+aqueous+solution.">Preparation and characterization of electrospun pullulan/montmorillonite nanofiber mats in aqueous solution.</a> Carbohydrate Polymers. 78 (2), 336-342 (2009).</li><li>Stijnman, A. C., Bodnar, I., Hans Tromp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+food-grade+polysaccharides.">Electrospinning of food-grade polysaccharides.</a> Food Hydrocolloids. 25 (5), 1393-1398 (2011).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+molecular+entanglements+in+starch+fiber+formation+by+electrospinning.">Role of molecular entanglements in starch fiber formation by electrospinning.</a> Biomacromolecules. 13 (8), 2247-2253 (2012).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+aspects+in+fabricating+pullulan+fibers+by+electro-wet-spinning.">Rheological aspects in fabricating pullulan fibers by electro-wet-spinning.</a> Food Hydrocolloids. 38, 220-226 (2014).</li><li>Han, 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=.">.</a> Fiber Spinning Rheology and Processing of Polymeric Materials: Volume 2: Polymer Processing. , 257-304 (2007).</li><li>Morris, E. R., Cutler, A. N., Ross-Murphy, S. B., Rees, D. A., Price, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+shear+rate+dependence+of+viscosity+in+random+coil+polysaccharide+solutions.">Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions.</a> Carbohydrate Polymers. 1 (1), 5-21 (1981).</li><li>Thompson, C. J., Chase, G. G., Yarin, A. L., Reneker, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+parameters+on+nanofiber+diameter+determined+from+electrospinning+model.">Effects of parameters on nanofiber diameter determined from electrospinning model.</a> Polymer. 48 (23), 6913-6922 (2007).</li></ol>

著者らは、開示することは何もないことを宣言します。

レオロジーは、従来の紡糸やエレクトロスピニング13を含むポリマーの加工を、研究するための不可欠なツールです。定常せん断レオロジー研究、ポリマーコンフォメーションと異なる溶媒でのそれらの相互作用から解決することができる（ 図2および3）。バイオポリマー分子が互いに重なるために十分ではない高濃度で、それらの濃度依存性は、良溶媒...

エレクトロスピニングは、多種多様な材料からナノスケールの繊維に連続したマイクロを生成することができる技術である。これは、増加し、学術および産業の関心1を得ています。セットアップとエレクトロスピニングの実施は簡単なように見えるが、繊維特性をelectrospinnabilityを予測し、制御する能力は、課題である。その理由は、多くのエレクトロスピニング工程2に影響を与える要因、プロセス、繊維が移動特にパスが存在するという事実にあるのかもしれない、カオス1である。しばしば経験的な「クック·アンド·ルック」アプローチは、潜在的electrospinnable物質をスクリーニングするために使用される。しかしながら、電界紡糸プロセス及び結果として得られる繊維の特性より良好な制御を得るために、electrospinnabilityを支配するメカニズムのより完全な理解が必要である。いくつかの研究者は、紡糸原液中のポリマーの分子の絡み合いは、エッセンであることを見出した成功したエレクトロスピニング3〜5はLの前提条件。 レオロジーは、ポリマー分散液中の分子の立体配座との相互作用を調査するための強力なツールです。例えば、マッキーら 。クロロホルム/ジメチルテレフタレート（7/3、v / v）を含む溶媒中で線形の分子配座および分枝状ポリ（エチレンテレフタレート - コ - エチレンテレフタレート）共重合体を調査し、ポリマー濃度が2-2.5xをする必要があったと判断さ成功したエレクトロスピニング4用の絡み合い濃度。 なぜなら、生分解性、生体適合性、および気力それらの合成対応更新性におけるそれらの利点のバイオポリマーからの繊維で現在新たな関心がある。しかし、専門家は、その構造の複雑さ、熱加工と劣る機械的性質の困難さから、一般的に発生する多くの課題に直面する。植物組織に見られるデンプンは、アモンですgの地球上で最も豊富で安価なバイオポリマー。最近6に記載された純粋な澱粉繊維は、電気湿式紡糸装置を使用して製造。プルランは、ある種の細菌によって細胞外に生産さ線状多糖類である。 （1→4）および（1→6） ​​の規則的な交互のグルコシド結合は機能7,8を形成する優れた繊維/フィルムを含むプルランのいくつかの独特の特性の原因であると考えられている。水性分散液からプルラン繊維の電界紡糸は、研究者9,10の数で報告されている。われわれの以前の出版物では、2つのバイオポリマー、デンプン11及びプルラン12のelectrospinnabilityは、議論されている。このレポートは、これらの2つの生体高分子のelectrospinnabilityの調査にレオロジーの原則を利用するためのプロトコルを示すに焦点を当てています。

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:253px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelose 80 starch</td>
			<td style="width:253px;">Ingredion</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pullulan</td>
			<td style="width:253px;">Hayashibara Co. Ltd</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:253px;">BDH Chemicals</td>
			<td style="width:119px;">BDH1115-4LP</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Ethanol</td>
			<td style="width:253px;">VWR International</td>
			<td style="width:119px;">89125-172</td>
			<td>200 proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:253px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rheometer</td>
			<td style="width:253px;">TA Instruments</td>
			<td style="width:119px;">ARES&#160;</td>
			<td>50 mm cone and plate geometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe (10 mL)</td>
			<td style="width:253px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">309604</td>
			<td>Syringe with Luer-Lok&#174; Tip</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">High voltage generator</td>
			<td style="width:253px;">Gamma High Voltage Research, Inc.</td>
			<td style="width:119px;">ES40P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pump</td>
			<td style="width:253px;">Hamilton Company</td>
			<td style="width:119px;">81620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">FEI Company</td>
			<td style="width:119px;">Quanta 200</td>
			<td>for starch fibers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">Phenom-World</td>
			<td style="width:119px;">Phenom G2 Pro</td>
			<td>for pullulan fibers</td>
		</tr>
	</tbody>
</table>

molecular entanglement and electrospinnability of biopolymers

エレクトロスピニングは、多種多様な材料からナノスケールの繊維にマイクロ作製する魅力的な技術である。バイオポリマーは、紡糸原液中の成分ポリマーの分子絡み合いが成功したエレクトロスピニングのための必須の前提条件であることが見出された。レオロジーは、生体高分子の分子コンフォメーションとの相互作用を調査するための強力なツールです。本稿では、それらのジメチルスルホキシド（DMSO）/水分散液から、2の生体高分子、デンプンおよびプルランのelectrospinnabilityを評価するためにレオロジーを利用するためのプロトコルを示しています。ミクロン範囲にサブミクロンの平均直径を持つ整形式デンプン及びプルラン繊維が得られた。 Electrospinnabilityが形成された繊維の視覚的な顕微鏡観察により評価した。彼らのelectrospinnabilityに分散液のレオロジー特性を相関させることによって、私たちは、分子の立体配座は、分子の絡み合い、せん断粘度はすべて当選者に影響を与えることを実証しているrospinning。レオロジーは、溶媒系の選択およびプロセスの最適化において有用であるだけでなく、分子レベルで繊維形成のメカニズムを理解していない。

バイオポリマーの濃度や溶媒中のDMSO濃度の関数としてのバイオポリマー分散液の流動曲線が得られた。二つの代表的な数値は、純粋なDMSO溶媒中のそれらの濃度の関数として澱粉（ 図2A）およびプルラン（ 図2B）の流動曲線を示す。具体的な粘度は、バイオポリマーの濃度（プルラン用デンプンおよび図3Bは図3A）に対してプロットした。こ?...

Watch this Scientific Journal Video about Molecular Entanglement and Electrospinnability of Biopolymers at JoVE.com

Molecular Entanglement and Electrospinnability of Biopolymers

エレクトロスピニングは、多種多様な材料からナノスケールの繊維にミクロを製造するために使用される魅力的な技術である。紡糸原液中の成分ポリマーの分子絡み合いが成功エレクトロスピニングのために必須である。私たちは、2つの生体高分子、デンプンおよびプルランのelectrospinnabilityを評価するためにレオロジーを利用するためのプロトコルを提示する。

生体高分子の分子エンタングルメントとElectrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

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Bioengineering

13.6K ビュー.  Pennsylvania State University. エレクトロスピニングは、多種多様な材料からナノスケールの繊維にミクロを製造するために使用される魅力的な技術である。紡糸原液中の成分ポリマーの分子絡み合いが成功エレクトロスピニングのために必須である。私たちは、2つの生体高分子、デンプンおよびプルランのelectrospinnabilityを評価するためにレオロジーを利用するためのプロトコルを提...

ビデオ: Molecular Entanglement and Electrospinnability of Biopolymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

生細胞における分子ローターの蛍光寿命イメージング

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

生物工学

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

DNA損傷の検出のための半人工分子機械とその使用のin vitroでの組立に

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

バイオマーカー探索のためのメソポーラスシリカ薄膜の低分子量蛋白質の濃縮

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

グライディングアッセイを用いて生体高分子の曲げ剛性の測定

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

アプリジア 単一ニューロンの電気生理学的・分子的解析のための神経節の準備

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

マイクロスケール渦アシストシーケンシャル分子送達用エレクトロポ

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

マルチモーダル分子イメージングアプリケーションのためのBiofunctionalizedプルシアンブルーナノ粒子

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

単層カーボンナノチューブ上のバイオミメティックポリマーとエンジニアリング分子認識

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

乾燥風-LCインターフェイス上Megamolecular生体高分子の自己統合するための方法

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

ヒト好中球群れの化学化学および分子解析のための生体微粒子マイクロアレイ

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...