この研究は国立科学財団の中国 (21474057 および 21604076) によって支えられました。

警告: 使用前に関連するすべての材料安全データ用紙 (MSDS) を参照してください。ヒューム フードと保護具 (保護メガネ、保護手袋、白衣、等) の使用を含む化学実験を行う際は、適切な安全対策を使用してください。プロトコル標準セル処理テクニック (殺菌、細胞回復、細胞継、細胞凍結、細胞染色、等) が必要です。
 1 準備のヒドロゲル <ol><li><ol><li><ol><li>PEG の事前乾燥を ベンズアルデヒドの合成終了・ ディ ・修飾ポリエチレング リコール (PEG DF) のポリマー重量 4.00 g ペグ (4,000 MW、1.00 モル)。丸底フラスコ (250 mL) にそれを転送し、トルエン (100 mL) を追加します 。
			注: 異なる分子量とその他のペグ ハイドロゲル形成のために働くことができますが、異なる剛性につながる</li>。
			<li>熱熱銃 (またはホット プレート 40 ° C 付近) によって軽度すべてのポリマーを溶解するためにソリューションです。すべてのポリマーは、溶解後は、蒸発器を使用して、すべての溶剤を削除します。乾燥のペグを高分子にするこのディゾルブとドライ プロセスを 2 回繰り返します 。
			注意: これは発煙のフード細心の注意を実行する必要があります。トルエンは可燃性</li>。
		</ol></li> <li>PEG のベンズアルデヒド終了反応 <ol><li>フラスコに電磁攪拌機及びテトラヒドロ フラン (THF、100 mL) を追加し、攪拌を使用してペグを解散。すべての固体を完全に解消するソリューション (0.90 g, 6.0 mmol) 4-carboxybenzaldehyde、4-ジメチルアミノピリジン (0.07 g、0.6 モル) を順に追加します</li>。
			<li>追加 N, N &#39;-ヒドロキシサクシンイミド (DCC, 1.25 g, 6.0 mmol) ソリューションには満ちているフラスコに無水 CaCl 2 乾燥管を追加。室温で攪拌しながら、反応を維持 (~ 20 ° C) 約 12 h. の</li>
		</ol></li> <li>後反応プロセス <ol><li>反応が完了したら、真空による溶液生成白色固体をフィルターで除外します。冷たいジエチル エーテル (500 mL) に白色の固形物を沈殿させる攪拌下の溶液を注ぐ。フィルターによりすべての白色固体を収集し、ヒューム フード中の固形物を乾燥します</li>。
			<li>再び白色固体を THF に溶解する、任意の不溶性の白色固体を除外、白色固形物を沈殿させる新鮮な冷たいジエチル エーテルに溶液を注ぐ、それを乾燥します。2 ~ 3 回、この手順を繰り返し、真空乾燥オーブン (20 ° C、0.1 mbar) 完全にそれらを乾燥するために白色の固体。最終製品として白い粉を収集: ベンズアルデヒド終了 DF ペグ</li>。
		</ol></li></ol></li> <li>ハイドロゲルの調製 <ol><li>チューブ (10 mL) で DF ペグ (0.11 g、0.028 ミリ モル 0.22 g、0.055 モル; 0.44 g、0.110 mmol) のさまざまな量の重量を量る脱イオン水 (5.0 mL) を追加して、支援する渦や磁性攪拌器使用 ポリマーを溶解します</li>。
		<li>リコール キトサン (0.495 g, x 10 -3 m モル 6.18) 管内 (50 mL)、脱イオン水 (15.0 mL) を溶解し、キトサン溶液 (3 wt %) を均質化するため、数分間渦を使用します</li>。
		<li>は、チューブ (2.0 mL) で順次リコール キトサン溶液 (0.2 mL、3 wt %) と DF PEG 溶液 (0.2 mL) を追加します。数分で均一フォーム ヒドロゲルへの解決策を混合するのに渦を使用します。表 1 異なる機械的強度のヒドロゲルの比に従ってください 。
		注: は、すでにヒドロゲルを形成するかどうかを決定するため管反転方法を使用します</li>。
	</ol></li> <li>レオロジー解析 <ol><li>パラレル鋼板回転レオメータのレオロジー解析を実施 (径: 20 mm)。ゲル化テストの下のプレートにリコール キトサン溶液 (0.2 mL、3 wt %) を広がってください。キトサン溶液の表面に均等に滴下 DF PEG 水溶液 (0.2 mL、2 wt %) を追加し、ピペットで手早く混ぜます。低いアッパー プレートをしてテストを開始</li>
		<li>ハイドロゲルの &#39; s 剛性テスト カット ハイドロゲル円 (直径: 20 mm) 貯蔵弾性率を測定し、(G &#39;) 1% ひずみ時の周波数解析に対する値。典型的な G &#39; 6.3 rad s 値 &#8722; 1 表 1 に示す 。
		注: はヒドロゲルの動的結合の安定化を確保するためハイドロゲル形成後 0.5 h ゲルのレオロジー解析を実施します</li>。
		<li>ヒドロゲルの &#39; s の自己 healable 特性テスト、切りにするハイドロゲルおよび整数部分に自動修復する作品が集まります。輪を作ってヒドロゲル部分で切って (径: 20 mm) レオロジー解析を行うレオメータの上に置くと</li>。
	</ol></li></ol> 2 ゲルの 3 d 細胞培養 <ol><li>ゲル ゲル溶液の調製 <ol><li>の細胞培養媒体 (RPMI 1640 年 2.0 で追加の重量を量る DF ペグ (0.44 g、0.11 ミリ モル) (4.0 mL)、生殖不能の管に。mL) と高分子溶液 (20 wt %) を取得する高分子を溶解するために渦や撹拌機を使用します。注射器 (10.0 mL) でソリューションを読み込むし、ミクロンの細菌保フィルター (0.22 μ m) を通すことで消毒します</li>。
		<li>はリコール キトサン (0.165 g, x 10 -3 m モル 2.06) 生殖不能の管 (15.0 mL) で重量を量る、細胞培養媒体 (RPMI 1640、4.0 mL)、そしてリコール キトサン溶液 (4.0 wt %) を取得する高分子を溶解するために渦を追加します。注射器 (10.0 mL) でソリューションを読み込むし、0.22 μ m 保細菌フィルターとフィルターします</li>。
	</ol></li> <li>ゲルの細胞培養 注意: 実行すべてのセルのティッシュ文化フードに関連する手順。生殖不能の技術の基本的な知識が期待されます。
	10% <ol><li>細胞懸濁液 <ol><li>RPMI 1640 培養 L929 細胞の調製培 FBS、ペトリネットの 5% ペニシリン (10 mL) 皿 (直径 10 cm)、37 ° C、5% CO 2 で孵化させなさいと。媒体は使用する前に毎日変更します</li>。
			<li>は、トリプシン (0.025 w/v %) および EDTA を含む PBS で L929 細胞を収穫 (0.01 %w/v)、遠心分離 (70 x g、5 分) と再 RPMI 1640 培地 (1.0 mL) で細胞を中断します。血液カウント ボードの標準操作を使用してセルのカウントを実行します。細胞濃度を調整する細胞を再停止 〜 3.75 × 10 6 セル/mL</li>。
		</ol></li> <li>ヒドロゲルの電池を封止材 <ol><li>L929 細胞懸濁液 (0.4 mL、10 6 セル mL -1 x 3.75) を混ぜて渦による管内 (4.0 mL) リコール キトサン溶液 (0.4 mL)。ピペットの共焦点シャーレの中央に L929/リコール キトサン溶液 (0.8 mL) (直径 2.0 cm)。同じ皿にピペットの DF PEG 溶液 (0.2 mL)、ソリューションをミックス、ハイドロゲル形成を誘導してゆっくりピペットします 。
			注: は、ペトリ皿を傾けることによってハイドロゲル形成を評価します</li>。
			<li>直接細胞培養、ゲルの上に RPMI 1640 培養媒体 (1.0 mL) の追加額を追加。インキュベーター (37 ° C、5% CO 2) に埋め込まれた携帯ハイドロゲル (1.0 mL、1.5 wt % グリコール キトサン、4.0 wt %df ペグ 1.5 × 10 6 セル mL -1) を入れ、毎日媒体を変更します。セル封止後日 1、3、5、および 7 にセルイメージ投射のための準備します</li>。
		</ol></li> <li>注入後ゲルの後 3 D 細胞培養の準備ロード携帯ハイドロゲル (1.0 mL ステップ 2.2.2 を参照) (10.0 mL、48 G 針) の注射器。ハイドロゲル フォーム後共焦点イメージング用シャーレにヒドロゲルをゆっくりと注入します。ヒドロゲルの上に培地 (1.0 mL) の追加量を追加し、それは毎日変更します。インキュベーター (37 ° C、5% CO 2) にペトリ皿を置くし、その後にイメージングのための準備します 。
		注意: を確認し、注射器の安全操作プロトコルに従ってください</li>。
	</ol></li> <li>細胞生存率解析 <ol><li>共焦点観察 <ol>
<li>は、2 回の PBS (1.0 mL) でゲルをすすいでください。フルオレセイン ・ ジアセテート (FDA、0.5 mL, 0.05 mg/mL) と propidium (PI、0.5 mL、0.08 mg/mL) はヨウ化ソリューションの 15 分のゲルを染色します。染色後、すべての溶剤を削除します</li>。
			<li>観察励起波長 488 の下共焦点顕微鏡を用いたゲル nm のライブを視覚化する nm と死者の 543 細胞、それぞれ。中の細胞の均一な分布を検証するゲルの 2 μ m 深さごとを通して z スタックを取る 。
			注意: FDA の汚れの生きているセル PI 汚れ死んだ細胞中です</li>。
		</ol></li> <li>チューブ (4.0 mL) に酢酸 (拍、3 v %、1.0 mL) 5 分のピペットでハイドロゲル (1.0 mL) が低下します。遠心分離機 (70 x g、5 分) による細胞を収集し、再 RPMI 1640 培 (1.0 mL) で細胞を中断します。細胞カウント ボードを数える血を使用してを実行します</li>。
	</ol>
	</li>
</ol>

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著者が明らかに何もありません。

このプロトコル (図 1) で発表したハイドロゲルは 2 つの主要なコンポーネント: 天然高分子グリコール キトサンおよび合成ベンズアルデヒド終了高分子ゲル化剤 DF ペグ、両方の生体適合性材料であります。DF ペグの合成は、ワンステップ改質反応を使用して提示されます。分子量 4,000 のペグは、溶解度、変更効率、ハイドロゲルの剛性の問題でこのプロトコルに選ば?...

ヒドロゲルは大量の水と柔らかい物性を架橋高分子材料と彼らは多くの生物医学応用1,2で使用されています。ゲルは、生体内で細胞の生理学的環境に非常に類似している柔らかく、ぬれた環境を提供できます。したがって、ヒドロゲルは 3 D 細胞培養3,4の最も人気のある足場になっています。2D ペトリ皿培養に比べると、3 D 細胞培養細胞外マトリックス (ECM) 模倣し、増殖・分化のための5のための組み立てる細胞の微小環境を提供する迅速に進んでいます。さらに、天然高分子を含むゲルは、細胞が増殖し、3を区別するための生体適合性と促進環境を提供できます。由来の合成高分子ヒドロゲルは動物由来タンパク質またはウイルスのような複雑な影響を排除する、そのシンプルかつ明確なコンポーネントに適しています。3 D 細胞培養のためのすべてのハイドロゲル候補では、中でゲルが容易に作製し、一貫性のあるプロパティを持つが常に優先されます。異なる研究要件に合わせてハイドロゲルのプロパティを調整する施設は、よく6として重要です。
3 D 細胞培養7のための汎用性の高いハイドロゲル プラットフォームとなる動的なイミン化学を使用してリコール キトサンによるハイドロゲルの安易な準備をご紹介します。このメソッドでは、よく知られているバイオ セーフティー リコール キトサンを使用ヒドロゲルのネットワークのフレームを確立します。そのアミノ基は、ヒドロゲル8crosslinkages として動的なイミン結合を形成する高分子ゲル化剤として終了ベンズアルデヒド ポリエチレングと反応しました。動的なイミン結合を形成し、機械的に調節可能な架橋ネットワーク9,10,11ヒドロゲルを endowing 周囲に可逆的かつ responsively を分解します。高含水、生体適合材料と調節可能な機械的強度のため、ハイドロゲルが正常に 3 D 細胞培養12,13L929 細胞の足場として適用されます。ここのプロトコルは、高分子ゲル化剤合成、ハイドロゲルの準備、セルの埋め込み、3 D 細胞培養など、手順を詳しく説明します。
ヒドロゲルはまたその動的なイミンの crosslinkages、その多様々 なバイオ刺激(酸/pH、ビタミン B6 誘導体ピリドキサール、タンパク質パパイン等)への応答を含む、ヒドロゲルへことができることを示すための他のいくつかの機能を示しています8生理学的条件下で分解を誘導しました。ヒドロゲルはまた自己 healable と注射、ヒドロゲル最小限の侵襲的な注入法で投与できる薬剤と細胞の配達14,15では優位します。ヒドロゲルは磁気、温度、pH 応答性など16,17を満たすことができるような特定のプロパティを獲得する互換性のある機能性添加剤や特定のあらかじめデザインされた高分子ゲル化剤を追加することによって、幅広い研究要件。これらのプロパティでは、注射をするハイドロゲルの潜在的な能力薬の細胞in vitroとin vivoの生物医学研究とアプリケーションの両方で複数のキャリアを明らかにします。

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Glycol chitosan</td>
			<td style="width:360px;">Wako Pure Chemical Industries</td>
			<td style="width:131px;">39280-86-9</td>
			<td style="width:385px;">90% degree of deacetylation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Carboxybenzaldehyde</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>619-66-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N, N&#39;-dicyclohexylcarbodiimide</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>538-75-0</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride anhydrous</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>10043-52-4</td>
			<td>96%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-dimethylamiopryidine</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>1122-58</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneglycol</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>5254-43-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrahydrofuran</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>109-99-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toluene</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>108-88-3</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl ether</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>60-29-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>64-19-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anhydrous CaCl2</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>10043-52-4</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>596-09-8</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodide&#160;</td>
			<td>Sigma</td>
			<td>25535-16-4</td>
			<td>94%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 culture media</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td></td>
			<td>0.25%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Solarbio</td>
			<td></td>
			<td>0.01 M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin streptomycin solution</td>
			<td>Hyclone</td>
			<td></td>
			<td>10,000 U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rheometer</td>
			<td>TA Instrument</td>
			<td></td>
			<td>AR-G2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>710-3channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td>NCTC clone 929; L cell, L929, derivative of Strain L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Evaporator</td>
			<td>EYELA</td>
			<td></td>
			<td>N-1100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 guage needle</td>
			<td>ShanghaiZhiyu Medical Material Co., LTD</td>
			<td></td>
			<td>48-guage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM3000 B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope software</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat gun</td>
			<td>Confu</td>
			<td></td>
			<td>KF-5843&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

プロトコルは、動的イミン化学を用いたキトサン ベースのゲルを準備する安易な効率的、かつ汎用性の高い手法を提案します。終了合成ベンズアルデヒド高分子ゲル化剤とグリコール キトサンの溶液を混合することによって、ハイドロゲルを用意し、ヒドロゲルは室温で数分間で効率的に得られます。グリコール キトサン、高分子ゲル化剤、含水量の様々 な比率によって異なるゲル化時間と剛性の多彩なゲルが得られます。破損して、その外観と係数、crosslinkages として動的なイミン結合の可逆性ヒドロゲルを回復できます。この自己 healable プロパティにより、注入処理後整数一括ハイドロゲルに絞りの部分から自己治癒があるので注射するハイドロゲルです。ヒドロゲルも多動的イミン結合の別の平衡状態のため多くの生物活性の刺激に応答するものです。このハイドロゲルが生体適合性として確認された、L929 マウス線維芽細胞が埋め込まれた次の標準的な手順と細胞の増殖は簡単に 3 D 細胞培養プロセスによって評価されました。ヒドロゲルは細胞のための 3 D 環境の生理学的な模倣は利益を得た別の研究のための調節可能なプラットフォームを提供できます。そのマルチ応答性、自己 healable と注射のプロパティと共に、ヒドロゲルは薬と将来の生物医学アプリケーションのセルに複数のキャリアとして可能性があります適用できます。

ゲルの調製とその 3 D 細胞培養上でこのプロトコルの図式は、図 1で提供しています。ハイドロゲルの内容と異なる機械的強さの準備率の情報は表 1のとおりです。ヒドロゲルの自己 healable とレオロジー特性図2 周波数テスト対貯蔵弾性率によるハイドロゲルの剛性をプレゼントします。細胞の共焦点画像とヒドロゲルの文化の日と携?...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

ここで動的イミン化学を用いたキトサン ベース注射ゲルの作製について述べる。ハイドロゲルの機械的強度と 3 D 細胞培養への応用を調整する方法が掲載されています。

キトサンを用いた注射ゲルおよび 3 D 細胞培養への応用の準備

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Research

JoVE Journal

Chemistry

18.9K ビュー.  Tsinghua University. ここで動的イミン化学を用いたキトサン ベース注射ゲルの作製について述べる。ハイドロゲルの機械的強度と 3 D 細胞培養への応用を調整する方法が掲載されています。

ビデオ: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

合成、細胞の配送および<em&gt;インビボ</em&gt;デンドリマーベースのpHセンサの応用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

化学

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

超高性能液体クロマトグラフィーによる組成解析のための細菌細胞壁の分離と調製

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

白リンの活性化カルボニル装飾カルベンの調製および使用

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

酵素的、化学（メチル化）と物理（質量分析、核磁気共鳴）技術を用いた植物の壁ヘテロキシランのシーケンシング

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

EOB-DTPAとそのののGa（III）錯体に関する研究<sup&gt; 68</sup&gt; Gaの放射性標識アナログ

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

C60/Graphene ハイブリッド ナノ構造の作製と評価

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

ナノダイアモンドおよびその低減銀ナノ粒子の生体 Polydopamine 表面改質

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes and determine the corresponding photochemical mechanism. Then, we describe a protocol to perform ring-opening metathesis polymerization (ROMP) in solution and in miniemulsion using this NHC-photogenerating system. To photogenerate IMes, a system comprising 2-isopropylthioxanthone (ITX) as the sensitizer and 1,3-dimesitylimidazolium tetraphenylborate (IMesH+BPh4-) as the protected form of NHC is employed. IMesH+BPh4- can be obtained in a single step by anion exchange between 1,3-dimesitylimidazolium chloride and sodium tetraphenylborate. A real-time steady-state photolysis setup is described, which hints that the photochemical reaction proceeds in two consecutive steps: 1) ITX triplet is photo-reduced by the borate anion and 2) subsequent proton transfer takes place from the imidazolium cation to produce the expected NHC IMes. Two separate characterization protocols are implemented. Firstly, CS2 is added to the reaction media to evidence the photogeneration of NHC through formation of the IMes-CS2 adduct. Secondly, the amount of NHC released in situ is quantified using acid-base titration. The use of this NHC photo-generating system for the ROMP of norbornene is also discussed. In solution, a photopolymerization experiment is conducted by mixing ITX, IMesH+BPh4-, [RuCl2(p-cymene)]2 and norbornene in CH2Cl2, then irradiating the solution in a UV reactor. In a dispersed medium, a monomer miniemulsion is first formed then irradiated inside an annular reactor to produce a stable poly(norbornene) latex.

We describe a protocol to photogenerate N-heterocyclic carbenes (NHCs) by UV irradiation of a 2-isopropylthioxanthone/imidazolium tetraphenylborate salt system. Methods to characterize the photoreleased NHC and elucidate the photochemical mechanism are proposed. The protocols for ring-opening metathesis photopolymerization in solution and miniemulsion illustrate the potential of this 2-component NHC photogenerating system.

N-複素環カルベンの生成: 光誘起開環メタセシス重合のアプリケーション

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

発砲残渣の推定検出のための新しい細菌性バイオセンサの調製と適用

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

その中透過電子顕微鏡におけるグラフェン支持マイクロウェル液体細胞の調製

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...