Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

注意：使用する前に、関連するすべての化学物質の安全性データシート（SDS）を参照してください。薬剤スクリーニング実験は、そのバルク対応物に比べて付加的な危険性を有することができるナノ材料の合成および取り扱いを伴います。工学的制御の使用（ヒュームフード）と個人用保護具（PPE、 例えば 、安全長ズボン、閉じたつま先の靴、耐薬品性手袋、安全ゴーグル）などの実験を通して実施することに必要なすべての管理措置を、確認してください。 薬物スクリーニングのための化学試薬の調製<ol><li>金のNC合成のための前駆体<ol><li>金（III）塩化物溶液の15mMのを準備するために超純水6.9 ml中に金（III）塩化物溶液（99.99％微量金属ベース、希HCl中30重量％）30mgを溶解します。注意：金塩化物溶液は、腐食性及び刺激性です。目や皮膚に直接接触を避けるために適切なPPEを着用してください。 </li><li>ディ超純水1ml中のHSAまたはBSAのssolve 74 mgの74ミリグラム/タンパク質ストック溶液のmlで調製しました。 </li></ol></li><li>薬液<ol><li>所望の薬剤の量、 例えば 、（a）は、イブプロフェンのために1.9 mgであり、（b）はワルファリン2.8 mgであり、（c）はフェニトイン2.3 mg及び450 mMのを準備するために20μlのDMSO中の（d）スルファニルアミド1.5 mgの溶かします薬剤原液の。 注：これらの薬物は疎水性であり、水中で難溶性を有するため、DMSOを溶媒として選択されます。 </li></ol></li><li>他の試薬<ol><li> NaOH溶液を1.5 Mを調製するために超純水10mlにNaOHペレットの600ミリグラムを溶解します。尿素溶液の20 Mを調製するために超純水2ml中の尿素の2.4グラムを溶解します。 </li></ol></li></ol>タンパク質鋳型金NCの2合成<ol><li> HSA-テンプレート金のNC（HSA-AU NCS） <ol><li>二つの別々の温度制御可能な磁気攪拌で、それぞれがその中にマイクロ磁気撹拌棒を含む、二枚のガラスバイアルを置きますRERS。 </li><li> 60℃に1磁気撹拌機の温度を設定し、「60°C」と、その上にガラスバイアルにラベルを付けます。他のマグネチックスターラーは、それの上にガラスバイアルは「RT」とラベル付けされた室温（RT）に維持されます。 </li><li>タンパク質テンプレート内部のAuイオンのカプセル化を可能にするために、HSA溶液200μl、超純水200μl、および（特に指定のない限り360 RPMとして設定）を一定に撹拌しながら各バイアルに塩化金（III）溶液200μlを追加します。 </li><li>金のNCを形成するHSAの還元能力を活性化し、0分として、反応時間を記録するために開始するように2分後、各バイアルをNaOH溶液20μlを加えます。 </li><li> 20分毎に、384ウェル黒色プレートに試料の各々からの溶液の50μLを描画し、マイクロプレートリーダーを用いて発光スペクトルを測定します。典型的なスキャン設定：λEX = 370nmで、λEMは 410 = - 850 nmのを。 </li><li> 100分後に磁気撹拌機を停止します。 </li><li>シンクに流水でガラスバイアルをクールダウン。注意：ガラスバイアルは、高温になっています。燃焼を避けるために、耐熱手袋を着用してください。 </li><li>各サンプルは、異なる温度条件でのAu NCの形成速度を獲得するためにすべての時間で光電子スペクトルをプロットします。 </li></ol></li><li> BSA-テンプレート金のNC（BSA-AU NCS） <ol><li>マグネチックスターラーの上にマイクロ磁気撹拌棒を含むガラスバイアルを置きます。室温などの温度のままにしておきます。 </li><li> BSA溶液の200μlを、尿素を200μl、および一定の撹拌下でガラスバイアル中の塩化金（III）溶液200μlを混ぜます。 </li><li> 2分後、金のNCを形成するために、BSAの還元能力を活性化し、0分として、反応時間を記録するために開始するのNaOH溶液20μlを加えます。毎時反応混合物の光電子スペクトルを測定します。 注：BSA-AuのNCの光電子スペクトルを測定し、あまり頻繁にBSA-AuのNCの生成速度が原因で、尿素の少ない有効に同じ温度でHSA-AUのそれよりも遅いため、タンパク質を展開します。 </li><li> 7時間後、磁気撹拌機を停止します。尿素変性によって金NCの形成速度を確認するためにすべての時間で光電子スペクトルをプロットします。 </li></ol></li></ol> 3.低分子薬物スクリーニング<ol><li> HSAの異なる小分子薬のスクリーン相対的結合親和性<ol><li>温度制御可能なマルチポイントマグネチックスターラーの上に、それぞれがその中に磁気攪拌棒を含む、5ガラスバイアルを置きます。 、すなわちイブプロフェン、各薬物について、それぞれa、b、c、dのようワルファリン、フェニトイン、およびスルファニルアミドをこれらのバイアルにラベルを付けます。コントロールとして、純粋なHSAと1にラベルを付けます。 </li><li>各バイアルに、HSAの200μLを加えます。対応する4つのバイアルに薬剤溶液を1μlを追加します。コントロールにDMSOを1μlを追加します。撹拌機のスイッチをオンにし、360に回転速度を設定回転数とは、HSAに結合する薬剤を完了させるために1時間インキュベートします。 </li><li> 1時間後、ミリQ水200μlと、一定に撹拌しながら各バイアルに塩化金（III）溶液200μlを添加します。 10分間、一定に撹拌しながら60℃に温度を設定します。各バイアルに1.5 M NaOHを20μlを添加して、0分として、反応時間を記録するために開始します。 </li><li>すぐに384ウェルブラックプレートに各バイアルから溶液50μlを描き、発光スペクトル（0分として表示結果）を測定します。各サンプルごとに10分の発光スペクトルを記録します。四つの異なる時点で少なくとも4つのスペクトルを収集し、それを0、10、20、および30分です。 </li><li>一貫した傾向で結果を得るために、3.1.4数回 - 繰り返して、3.1.1を繰り返します。磁気撹拌機を停止します。 </li><li> （ - D、およびコントロール）各サンプルの時間分解光電子スペクトルをプロットします。ディに金NCの形成速度を比較するために、時間に対するすべてのサンプルとプロットのピーク強度を特定fferent薬物負荷タンパク質テンプレート。 </li></ol></li><li> HSAに特異的な薬物の結合定数を測定します<ol><li> 3.1.4 - を繰り返して、3.1.1を繰り返します。 （唯一の薬物負荷なしでHSAを使用して、コントロールサンプルを含む）は、4つの異なる濃度でイブプロフェンソリューションと薬液を交換してください。それに応じて薬物濃度に応じてガラスバイアルにラベルを付けます。 </li><li> 10分後、流しに流水でガラスバイアルをクールダウン。注意：ガラスバイアルは、高温になっています。燃焼を避けるために、耐熱手袋を着用してください。 </li><li> 384ウェルブラックプレートに各バイアルから上記の溶液の50μLを描画し、発光スペクトルを測定します。 </li><li>異なる薬剤濃度での結果の他の二組を得るために、3.2.3回以上 - を繰り返して、3.2.1を繰り返します。 </li><li>磁気撹拌機を停止します。光電子スペクトルをプロットし、その結果を分析します。 <ol><li>各個別のバッチで得られた生データの場合、各々の光電子スペクトルをプロットソフトウェアのサンプルなどOriginProソフトウェアなどとのピーク強度を見つけます。 </li><li> [ -私は0 /（I I 0）]私と私はそれぞれ、薬物負荷HSAと純粋なHSAに形成されたAu NCの蛍光強度を参照してください0薬物濃度、に対する相対蛍光強度を計算し、プロットします。 </li><li> 、C /（K D + C）×Y = R maxをR maxは最大応答信号を示し、Cは、リガンドの濃度である：ミカエリス-メンテンの式を使用して単一部位結合モデルにデータをフィットさせることにより結合定数を計算しますそしてK Dは、結合定数です。このようOriginProなどのソフトウェアでこれを実行します。非線形曲線フィットをフィッティングメニュー分析を選択し、 成長/シグモイドカテゴリからヒル機能を選択します。セッティング：機能の選択]ページ、フィット結果を表示するために合わせる]をクリックします。 </li></ol></li></ol></li></ol>

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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-Assisted+Synthesis+of+BSA-Protected+Small+Gold+Nanoclusters+and+Their+Fluorescence-Enhanced+Sensing+of+Silver(I)+Ions.">Microwave-Assisted Synthesis of BSA-Protected Small Gold Nanoclusters and Their Fluorescence-Enhanced Sensing of Silver(I) Ions.</a> Nanoscal. 4, 2251-2254 (2012).</li><li>Liu, Y., Ai, K., Cheng, X., Huo, L., Lu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanocluster+Based+Fluorescent+Sensors+for+Highly+Sensitive+and+Selective+Detection+of+Cyanide+in+Water.">Gold Nanocluster Based Fluorescent Sensors for Highly Sensitive and Selective Detection of Cyanide in Water.</a> 20, 951-1907 (2010).</li><li>Liu, J., Yu, M., Zhou, C., Yang, S., Ning, X., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+Tumor+Targeting+of+Renal-Clearable+Luminescent+Gold+Nanoparticles:+Long+Tumor+Retention+and+Fast+Normal+Tissue+Clearance.">Passive Tumor Targeting of Renal-Clearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance.</a> J. Am. Chem. Soc. 135, 4978-4981 (2013).</li><li>Negishi, Y., 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=Controlled+Loading+of+Small+Aun+Clusters+(n+=+10&#8211;39)+onto+BaLa4Ti4O15+Photocatalysts:+Toward+an+Understanding+of+Size+Effect+of+Co-Catalyst+on+Water+Splitting+Photocatalytic+Activity.">Controlled Loading of Small Aun Clusters (n = 10&#8211;39) onto BaLa4Ti4O15 Photocatalysts: Toward an Understanding of Size Effect of Co-Catalyst on Water Splitting Photocatalytic Activity.</a> J. Phys. Chem. C. , (2015).</li><li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoclusters.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters.</a> J. Am. Chem. Soc. 131, 888-889 (2009).</li><li>Celej, M. S., Montich, G. G., Fidelio, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Stability+Induced+by+Ligand+Binding+Correlates+with+Changes+in+Protein+Flexibility.">Protein Stability Induced by Ligand Binding Correlates with Changes in Protein Flexibility.</a> Protein Sci. 12, 1496-1506 (2003).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Analysis+of+Ligand-Induced+Changes+in+Protein+Thermal+Unfolding+Applied+to+High-Throughput+Determination+of+Ligand+Affinities+with+Extrinsic+Fluorescent+Dyes.">Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes.</a> Biochemistr. 49, 10831-10841 (2010).</li><li>Yu, Y., New, S. Y., Xie, J., Su, X., Tan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Based+Fluorescent+Metal+Nanoclusters+for+Small+Molecular+Drug+Screening.">Protein-Based Fluorescent Metal Nanoclusters for Small Molecular Drug Screening.</a> Chem. Commun. 50, 13805-13808 (2014).</li><li>Shortridge, M. 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> Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and Drug Discover. , (2010).</li></ol>

The authors have nothing to disclose.

この方法で強調表示される必要があるいくつかの重要なステップがあります。別の小分子薬、ステップ3.1.2、3.1.3、および3.1.4の相対的結合親和性をスクリーニングするプロトコルでは相対的な結合強度のための一貫した傾向を示す良い結果を得るために重要です。これらの手順では、化学物質を添加して測定用反応液を描画するアクションは、時間遅れ効果と化学薬品を添加して測定用の反?...

例えば、ヒト血清アルブミン（HSA）およびウシ血清アルブミン（BSA）などの血清アルブミンは血漿中で最も豊富なタンパク質であり、血液区画の浸透圧の維持に重要な役割を演じます。それらはまた、ステロイド、脂肪酸、甲状腺ホルモン、および薬物の広範囲のような低水溶性の小分子のための担体タンパク質として認識されています。アルブミン血清するために、これらの分子の結合特性（例えば、結合部位は、結合親和性や強度）は、薬物動態における重要なトピックとなっている。1-4、いくつかの分析方法は、アルブミン、血清するために、異なる薬剤の結合特性を研究するために開発された、などX線結晶学、5,6-核磁気共鳴（NMR）、7-11、および表面プラズモン共鳴（SPR）、12,13等しかしながら、これらの方法は、面倒で時間のかかる解 ​​析処理（どちらかによって制約され、例えば 、X線crystalloための単結晶の成長グラフィック研究）、特殊で高価な機器の要件（SPR）、または検出のための高価な同位体標識（NMR）を必要としました。これは、高速、単純で、かつコスト効率的な方法で低分子薬物スクリーニングのための別の方法を開発することが非常に望ましいです。 金ナノクラスター（金のNC）が2ナノメートルよりも小さいサイズの金属原子の十を含有する、ナノ材料の特殊なタイプです。14-17彼らは、ディスクリートおよびサイズ依存電子構造に起因する大規模な研究関心を集めている、18、 19と分子状の吸収と排出。20-23そのような固有の材料特性は、特に、強い蛍光は、生体系におけるそのような感知およびイメージングのような多様な用途が見出されている。24-32超小型蛍光金のNCは、機能性タンパク質を使用して合成することができますこのようなテンプレートとして血清アルブミン、など。33の典型的なタンパク質-テンプレート合成で<html>金NCS、金塩の一定量の第一のタンパク質の内部にカプセル化され、続いてタンパク質自体によって低減されます。タンパク質の還元能力は、アルカリ性溶液のpHを増加させることによって活性化することができる機能性アミノ酸残基（例えば、チロシン）の構成要素に起因します。タンパク質構造のアンフォールディングは、金NCの形成のための重要なステップであると考えられます。折り畳まれていないタンパク質に、より多くの還元性官能基は、カプセル化された金塩に曝露することができるからです。アンフォールディングタンパク質は、熱処理または変性剤に曝露することによって達成することができます。小分子薬物はまたすなわち中点変性温度と、これらすべての要因の。34,35効果展開のエンタルピーを変更する、展開プロセスに影響を与える可能性の導入は、次に、蛍光金NCの形成速度によって反映させることができるとで明らかに結果として得られる金NCの蛍光強度。36 e_content &quot;&gt;このビデオは、より高い温度（60℃）で、または変性剤の存在下で薬物負荷アルブミンタンパク質で金のNCを合成することにより、薬剤スクリーニング方法（例えば、尿素）、得られたAu NCの蛍光強度を示しています信号読み出しである。まず、金のNCは、金のNC。第二の生成速度に影響を与えるタンパク質は（熱処理または変性剤によって誘導される）アンフォールディングする方法を示すために、60℃で、または尿素の存在下で処理したHSAとBSAテンプレートに合成され、金のNCは、異なる薬物でプリロードタンパク質鋳型で合成され、得られたAu NCの相対蛍光強度に対する薬物負荷効​​果は相対的結合強度の尺度を提供する、研究されている。最後に、金NC-薬物スクリーニングプロトコルのために修正されます一定濃度のタンパク質にプリロード薬物含有量を変化させることにより、薬物-タンパク質結合定数（K D）の定量的測定。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

我々は、AuのNCによって放出された差動蛍光信号に基づいて、薬物を装填タンパク質内金（Au NCS）蛍光金ナノクラスターを形成することにより、標的タンパク質に小薬物分子の結合親和性を決定するための新薬のスクリーニング方法を実証します。例えば、ヒト血清アルブミン（HSA）およびウシ血清アルブミン（BSA）などのアルブミンタンパク質は、モデルタンパク質として選択されます。アルブミンタンパク質に対する異なる結合親和性の四つの小分子薬 （例えば、イブプロフェン、ワルファリン、フェニトイン、およびスルファニルアミド）がテストされます。これは、変性条件（ すなわち 、60°Cまたは尿素の存在下）下で薬物ロードアルブミンタンパク質内部の蛍光金NCの形成速度を（薬物なし）自然のままのタンパク質内に形成されたものよりも遅いことが判明しました。また、として形成されたNCの蛍光強度は逆に、アルブミンタンパク質に対するこれらの薬剤の結合親和性に相関することが見出されています。特に、金のNC形成の速度より遅い、薬物 - タンパク質結合親和性より高い、したがって、結果として得られる金NCの低い蛍光強度が観察されます。結果として得られる金NCの蛍光強度は、したがって、テストした異なる薬剤の相対的な結合強度の単純な尺度を提供します。この方法は、単に一定のタンパク質濃度でタンパク質にプリロード薬物含有量を変化させることによって、特異的な薬物-タンパク質結合定数（K D）を測定するために拡張可能です。測定結果は、他の威信が、より複雑な方法を用いて得られた値とよく一致します。

タンパク質の多くの反応性官能基 （例えば、チロシン残基）がカプセル化された金イオンを還元し、こうして金NCの形成速度を加速するために露出させることができるので、アンフォールディングタンパク質は、タンパク質鋳型金NCの形成のための重要な手順です。加熱および外部変性剤には、タンパク質のアンフォールディングプロセスを促進するための2つの一般的な手段である。 ...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

タンパク質 - 小分子標的薬のスクリーニングのための迅速かつ定量的蛍光法

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

Research

JoVE Journal

Bioengineering

9.9K ビュー.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

ビデオ: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

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

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

マイクロパターン細胞を用いた薬物の効果の改善可視化と定量分析

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

生物工学

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

ハイスループット抗真菌薬スクリーニングのためのカンジダバイオチップ（ カルシウム BChip）

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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

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

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

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

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

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

バイオメディカル応用のためのバイオインスパイアードナノハイドロキシアパタイトの急速なミックス準備

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

心臓医薬品安全性スクリーニングのための人間エンジニア心臓組織の自動収縮分析

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

注射の再生療法と高スループットの薬剤のスクリーニングのための 3 D への放射能

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

化学のエピジェネティックな修飾剤の細胞機械装置をリダイレクトすることによって遺伝子の転写を抑制

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

自動化された微生物叢システムを用いた中国ハムスター卵巣細胞からのモノクローナル抗体の精製と分析

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

オープンソースマイクロプレート互換照明パネルのアプリケーション

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...