The authors would like to convey their gratitude for the technical support provided by National Synchrotron Radiation Research Center (NSRRC) (Taiwan). The authors are grateful for the financial support provided by the Ministry of Science and Technology of the Republic of China (Taiwan) and the Industrial Technology Research Institute (Taiwan).

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The authors have nothing to disclose.

双極子支援SPEのマイクロチップの製造のための詳細な手順は、上記に提示しました。このセクションでは、PMMAのC-Clで部分および微量金属イオンを決意するための抽出媒体として使用されたのCl含有PMMAの可能性の移植に関して修正プロトコルの有用性は、ありますステップバイステップを評価しました。表面の検証のために、試料の種類は分析機器との適合性に基づいて選択しました。換言すれば、同様の工程を経て調製された試験サンプルの種類は、分析機器の要件に従って決定しました。例えば、粉末充填型のサンプルは、LA-ICP-MS、ラマン分光に使用したのに対し、基板型サンプルは、接触角の測定に使用し、XANES解析します。 最初は、化学官能アタッシュケースが受ける変化を監視します提案されている手順の間PMMAの表面にD、各工程を行ったに対応する得られた生成物に対する接触角分析（ 図2）。 図2に表示されているように、接触角の変化は明らかに表面変化は、修正手順の間に発生したことが示され、そして最終生成物を測定したところ80.3°±0.43°の接触角は、以前の結果を報告した。21と一致しましたまた、変性PMMAのC-Clでの成分の存在はまた、LA-ICP-MS分析によって確認しました。ネイティブPMMAをアブレーションすることによって得られた結果と比較して、CLには別個の信号はC-Clで部分で修飾されたPMMAをアブレーションすることによってexpectably観察された（ 図3（a）参照 ）。 ラマンスペクトルは、さらに、PMMAのC-Clで部分の結合を検証するために収集しました。 Figuに示すように図3（b）再もしくはCCl 2非対称伸縮振動に関連する2つの特徴的なピーク682 cm -1で718 cm -1で修正されたPMMAのスペクトル中で観察したウィリスらによって報告された結果とのことで適度に良好な一致22、ヘンドラら 23言い換えると、PMMAのC-Clでの部分の結合が正常に変更した後に達成され得ます。 また、本研究で提案された抽出機構を解明するために、XANES解析を使用しました。 図4に示されるように、高度に電気陰性のC-Clで部分と正に帯電した金属イオンとの間の相互作用は、MN 2+イオンで ​​処理された変性PMMAに対応XANESスペクトルにおける優勢な吸収端が存在することによって確認することができました。このように、双極子静電相互作用は確かTRのためのオンチップ抽出に適用されますエースの金属分析します。台湾の二つの川から採取した水サンプルの詳細な分析結果を他の場所に記載されている。16 我々の知る限り、これは微量金属イオンの決意のためのオンチップSPE反応で革新的な作業戦略を利用する最初の試みであり、開発したデバイスは、他のオンチップSPE技術と比較して有意に耐久性であったこと（ すなわち、 、160以上の分析作品）が、抽出効率の点で大幅な劣化なしに達成することができます。そのような抽出機構は、主として高度に電気陰性のC-Clで部分と正に帯電した金属イオンとの間の相互作用に依存しているため、それにもかかわらず、提案された技術は、これまで負に帯電した種を抽出するには不向きであることが予想されました。

環境管理と汚染防止の観点から、深刻な汚染や毒物学的問題を引き起こす微量金属は、世界的に懸念されます。生試料中の予想外の共存化学種は、多くの場合、トレースの量で存在する分析物の正確な決意を妨げるため、適切なオンチップ・サンプル前処理技術が広く、処理の成功への鍵とチップベースのプラットフォームを介して、実際のサンプルを分析することとして受け入れられています。利用可能な技術のうち1、オンチップ固相抽出（SPE）微量金属のために特に人気が同時に実行される試料のクリーンアップおよび検体前濃縮を可能にするこの技術は、複雑な塩マトリックスからの金属イオンの単離のために非常に有用であるので、分析。 2,3 微量金属の決意に使用されるオンチップSPE技術の進歩は着実に進化してきました。初期の頃には、トン彼はチップを樹脂充填SPEユニットを構成するためにマイクロチャネルに市販の樹脂をロードすることによって調製したSPE 4-7これは時折、樹脂保持可能な形態への金属イオンの変換を可能にするように誘導体化される検体を必要とした。4代替方法チップベースのSPEデバイスの製造のための単純な表面改質後の微量金属の回収のためのSPEの吸着剤としてのチップチャネルを利用することである。8近年、磁性ナノ粒子（MNPの）および特定の化学物質の取り込みを含む新興の傾向を見てきました金属イオンを効率的に保持することができる官能基を含有します。商業的な樹脂とは対照的に、のMNPは、それらが外部磁界Tの助けを借りてマイクロチャンネルに充填された後にγメルカプトプロピルトリメトキシシラン（γ-MPTS）9とアミノベンジルエチレンジアミン四酢酸（ABEDTA）10のような化合物で修飾されていますO金属イオンの選択的抽出を実現します。 オンチップSPE技術の開発における重要な進歩を目撃してきたが、報告された技術は、典型的には、イオン交換またはキレート化のいずれかに基づいて機能します。このような技術の使用は、分析性能を維持するためにエアコン、洗濯、又は再生に関連するものを含む不可避操作手順を必要とするという欠点があります。残念ながら、追加の操作手順の必要性は、各分析に必要な時間を延長するだけでなく、高いブランク値と再現性のない結果を引き起こす危険性があります。11のため、オンチップのSPE技術のための戦略を働い代替は微量金属分析のために不可欠です。 1993年に、ワットChehimi 12は、金属イオンは、ポリマー材料に向けて保持する傾向を有することを見出し、そして分析物のほとんどは、効率的に、クロロ（CL）上に保持されていることを-containingのポリマー材料ナトリウムイオンを除いて、ポリ（塩化ビニル）（PVC）。そのため、2002年に、Eboatu ら 13は、さらにPVCによってソリューションからいくつかの有毒金属の隔離について報告しました。これは、検体の前濃縮および塩マトリックスの除去のための優れた特性を示した高分子材料をClを含有することを示したので、のCl含有SPE機能を備えたチップベースのデバイスは、決意のための新規のオンチップSPE技術の開発のための魅力的な戦略と考えられました微量金属イオン。そのような製造の容易さ、所望の化学的/機械的特性、および光学的透明性などを考慮すると材料の機能は、14,15この研究では、マイクロデバイスを製造するために、ポリ（メチルメタクリレート）（PMMA）を利用しました。そして、CL-含有SPE機能は、微量金属イオンの決意するための新規なオンチップSPE技術の開発のために作製した素子に注入した。16 Remarkably、チャネル内部の高度に電気陰性C-Clでの部分の間の双極子 - イオン相互作用および正に帯電した金属イオン上の革新的な抽出機構の依存はにつながる、一般的なオンチップのSPE手順の間に講じた措置を回避することができます過剰の試薬を使用することによって引き起こされる汚染や追加のステップに起因する労働者のいずれかの劇的な低下。この貢献で提供されたプロトコルは、自分の仕事のための双極子支援SPEのマイクロチップを製造するために多様な背景から研究者を可能にします。製造されたマイクロチップのための詳細な特徴付け手順も同様に記載されています。

<table border="0" cellpadding="0" cellspacing="0" width="1311">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">AutoCAD</td>
			<td style="width:231px;">Autodesk</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">http://www.autodesk.com/education/free-software/autocad</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Poly(methyl methacrylate) (PMMA) sheet</td>
			<td style="width:231px;">Kun Quan Engineering Plastics</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">350 mm (L) x 20 mm (W) x 2 mm (H). The glass transition temperature (Tg) of PMMA sheets is ranged from 102&#8211;110 &#176;C. The UV transmittance of the PMMA at 365 nm is 91.2%.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:383px;">Micromachining system</td>
			<td style="width:231px;">Laser Life</td>
			<td>LES-10</td>
			<td style="width:423px;">Maximum laser power: 10 W. Maximium engraving speed: 762 mm s&#8722;1.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">High-resolution optical microscope</td>
			<td style="width:231px;">Ching Hsing Computer-Tech</td>
			<td style="width:276px;">FS-230</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Power Image Analysis system (PIA)</td>
			<td style="width:231px;">Ching Hsing Computer-Tech</td>
			<td style="width:276px;">PIA V16.1</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Multi drilling machines</td>
			<td style="width:231px;">N/A</td>
			<td>LT-848</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Deionized water (D. I. H2O)</td>
			<td style="width:231px;">Millipore</td>
			<td>Milli-Q Integral 5 System</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:231px;">J. T. Baker</td>
			<td>4095-04</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Ultrasound oscillator</td>
			<td style="width:231px;">Elma</td>
			<td>Transsonic Digital</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Glass board</td>
			<td style="width:231px;">N/A</td>
			<td style="width:276px;">N/A</td>
			<td>160 mm (L) x 35 mm (W) x 2 mm (H); fragile</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;">Binder clip</td>
			<td style="width:231px;">SDI</td>
			<td style="width:276px;">0234T-1</td>
			<td style="width:423px;">http://stationery.sdi.com.tw/product_detail.php?Key=322&#38;cID=55&#38;uID=6</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Precision oven</td>
			<td style="width:231px;">Yeong Shin</td>
			<td style="width:276px;">DK-45</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Poly(etheretherketone) (PEEK) tube</td>
			<td style="width:231px;">VICI</td>
			<td style="width:276px;">JR-T-6002 (0.5 mm i.d.); JR-T-6001 (0.25 mm i.d.)</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Polymer tubing&#160; cutter</td>
			<td style="width:231px;">Upchurch Scientific</td>
			<td style="width:276px;">A-327</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:53px;">Two-component epoxy-based adhesive</td>
			<td style="width:231px;">Richwang</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">Skin irritative. The major components are an epoxy resin and a hardener.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Peristaltic pump</td>
			<td style="width:231px;">Gilson</td>
			<td>Minipuls 3</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Peristaltic tube</td>
			<td style="width:231px;">Gilson</td>
			<td>F117934</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Sodium hydroxide (NaOH)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">30620</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Nitric acid (HNO3)</td>
			<td style="width:231px;">J. T. Baker</td>
			<td style="width:276px;">959834</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Acrylamide (prop-2-enamide, C3H5NO)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">A8887</td>
			<td>Acutely toxic and carcinogenic</td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:383px;">In-house-built photomask</td>
			<td style="width:231px;">N/A</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">The in-house-built photomask was made of a black paper (114 mm (L) &#215; 22 mm (W)) that contained an open window (94 mm (L) &#215; 2 mm (W)) allowing the desired region</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">1,1-Dichloroethylene</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">163032</td>
			<td>Acutely toxic and carcinogenic</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Cartridge</td>
			<td style="width:231px;">Dikma</td>
			<td style="width:276px;">ProElut AL-B&#160;</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">2,2-Azobisisobutyronitrile (AIBN, C8H12N4)</td>
			<td style="width:231px;">Showa Chemical&#160;</td>
			<td style="width:276px;">0159-2130</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Ethanol</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td>32221</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Hexanes (C6H14)</td>
			<td style="width:231px;">Millinckrodt Chemical</td>
			<td style="width:276px;">5189-08</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:383px;">&#160;In-house-built irradiation system</td>
			<td style="width:231px;">Great Lighting (UV-A lamp)</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">An opaque box with an UV-A lamp (40 W, maximum emission at 365 nm)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Glass vial</td>
			<td style="width:231px;">Yeong Shin</td>
			<td style="width:276px;">132300019</td>
			<td>Fragile</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Aluminum foil</td>
			<td style="width:231px;">Diamond</td>
			<td style="width:276px;">N/A</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Conical tubes with screw caps</td>
			<td style="width:231px;">labcon</td>
			<td style="width:276px;">3181-345-008 (50 mL); 3131-345-008 (15 mL)</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Rocking shaker</td>
			<td style="width:231px;">TKS</td>
			<td style="width:276px;">RS-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Contact angle meter</td>
			<td style="width:231px;">First Ten Angstroms</td>
			<td>FTA 125</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">PMMA bead</td>
			<td style="width:231px;">Scientific Polymer Products</td>
			<td style="width:276px;">037A</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Mortar and pestle, agate&#160;</td>
			<td style="width:231px;">Yeong Shin</td>
			<td style="width:276px;">139000004</td>
			<td>Fragile</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Tissue culture plate</td>
			<td style="width:231px;">AdvanGene Life Science Plasticware</td>
			<td style="width:276px;">AGC-CP-24S-50EA</td>
			<td>24-Well, non-treated, sterilized</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Hydraulic press</td>
			<td style="width:231px;">Panchum</td>
			<td style="width:276px;">Press-200</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Laser ablation</td>
			<td style="width:231px;">New Wave Research</td>
			<td>NWR193</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:383px;">Inductively coupled plasma-mass spectrometer</td>
			<td style="width:231px;">Agilent Technologies</td>
			<td>Agilent 7500a</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:383px;">Glass bottle</td>
			<td style="width:231px;">DURAN</td>
			<td style="width:276px;">21801245 (100 mL); 21801365 (250 mL)</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Dispersive Raman spectrometer</td>
			<td style="width:231px;">Thermo Fisher Scientific</td>
			<td>Nicolet Almega XR</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Manganese nitrate tetrahydrate (Mn(NO3)2&#215;4H2O)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">63547</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Maleic acid disodium salt hydrate (C4H4Na2O5)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">M9009</td>
			<td></td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:383px;">X-ray absorption near edge structure (XANES)&#160;</td>
			<td style="width:231px;">N/A</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">The Mn K-edge XANES analyses were conducted at 07A and 17C1 beamlines of the National Synchrotron Radiation Research Center (NSRRC) in Taiwan.</td>
		</tr>
	</tbody>
</table>

fabrication of a dipole-assisted solid phase extraction microchip for trace metal analysis in water samples

本論文では、水試料中の微量金属分析のために利用可能な双極子支援固相抽出（SPE）マイクロチップの製造プロトコルについて説明します。チップベースのSPE技術の進化の概要が提供されます。これは、特定のポリマー材料への導入とSPEにおけるその役割が続いています。革新的な双極子補助SPE法を開発するために、SPE機能を含有塩素（CL）は、ポリ（メチルメタクリレート）（PMMA）マイクロチップに注入しました。ここで、接触角分析、ラマン分光分析、およびレーザーアブレーション、誘導結合プラズマ質量分析法を含む様々な分析技術（LA-ICP-MS）分析は、上のC-Clで部分の注入プロトコルの有用性を検証するために使用しましたPMMA。 X線吸収端近傍構造解析結果（XANES）分析もまた、双極子によって抽出媒体としてのCl含有PMMAの実現可能性を実証しました高度に電気陰性C-Clで部分と正に帯電した金属イオンとイオンの相互作用。

図2は、PMMAマイクロチップのチャネル変更の手続きの際に発生する反応を示しています。接触角分析は、提案されている手順の間、表面の変更を監視するために使用しました。 LA-ICP-MSシステムおよび分散型ラマン分光計はC-Clでの成功した変更を確認するために使用されたがPMMA基板上に形成する部分（ 図3の（a）、（b）参照 ）。提案された双極子補助SPE反応はXANES解析（ 図4）によって特徴付けました。 <img alt="図1" src="/files/ftp_upload/53500/53500fig1.jpg" /> 図1. PMMAマイクロチップ。（a） の製作マイクロチップ用のパターンファイルのスナップショット。 （b）は製作マイクロチップのレイアウト：S、E、およびBは、試料のための導入ポートを表し、溶離液、およびBUFをそれぞれの溶液をFER。 Oは、出口を表しています。黒丸はそれぞれのための掘削のアクセスホールを表します。サンプルおよびバッファソリューションの導入に使用されるチャネルの両方が抽出チャネルと30°の角度を形成しました。コンフルエント出口に試料と緩衝液の流れの収束点からの距離とした効果的な抽出流路の長さは94 mmでした。 （c）は 、機械加工板の断面写真。文献から再生。 16化学の王立協会の許可を得て。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53500/53500fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53500/53500fig2.jpg" /> PMMAマイクロチップのためのチャネル変更の 図2. スキーム。差し込みphotogrAPHSシーケンスで得られた生成物に対応する接触角を示します。接触角は、水滴の画像を用いて決定しました。 3回の繰り返し測定の平均は、それぞれの場合において報告接触角を測定するために使用しました。文献から再生。 16化学の王立協会の許可を得て。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53500/53500fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53500/53500fig3.jpg" /> PMMAの変更の 図3. 表面検証。（A）のClのための信号がC-Clでの部分で修飾されたPMMA及びPMMAの両方を切除することにより得られます。挿入図は、各取得信号に対応する切除位置を示しています。 （b）は 、ネイティブのラマンスペクトルとPMMAを変更しました。文献から再生。の許可を得て16化学の王立協会。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53500/53500fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53500/53500fig4.jpg" /> 修正されたPMMA及びMn 2+ イオン で ​​処理された修正されたPMMAの 図4 のMnのK端XANESスペクトル 。修正されたPMMAのスペクトルは赤線のように発表されました。吸収スペクトルを示す変形PMMAの高度に電気陰性C-Clで部分とMn 2+イオンとの間の相互作用は、青色の線として提示されました。文献から再生。 16化学の王立協会の許可を得て。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53500/53500fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples at JoVE.com

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

水試料中の微量金属分析のためのダイポールアシスト固相抽出マイクロチップの作製

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

fabrication-dipole-assisted-solid-phase-extraction-microchip-for

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8.6K ビュー.  National Tsing Hua University. The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

ビデオ: Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

核酸、タンパク質と低分子のReagentless検出用電気化学DNAバイオセンサーの作製

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

生物工学

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

高秩序のナノシートにペプトイドポリマーおよびそれらの自己組織化の固相Submonomer合成

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

バイオメディカル応用のための機械的に調整可能な生物活性金属スキャフォールドの作製

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

強化された暗視野顕微鏡とハイパースペクトルマッピングにより組織学的試料中の金属酸化物ナノ粒子の同定

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

限外濾過/ナノ濾過膜性能試験のための三次元プリントマイクロ流体クロスフローシステム

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

シトクロムをカプセル化<em&gt; C</em&gt;金属ナノ粒子なしのシリカエアロジェルナノアーキテクチャで気相生物活性を保持しながら、

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中のパルスレーザアブレーションによる金属ナノ粒子の生成：ナノ粒子の抗菌特性を調べるためのツール

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...