親切に図5に示すデータの収集と分析を支援し、マイケル·ヘンリー、ダブリン市立大学、。この作品は、（1）ジョージ·メイソン大学によって部分的にサポートされていた、イタリア/アメリカの枠組みの中で（2）イタリア語IstitutoSuperioreディ·サニ&#39;米国保健社会福祉省、ジョージ·メイソン大学、公衆衛生のイタリア省、LAL〜（3）のNIH、IMATプログラム助成1R21CA137706-01と1R33CA173359-01、および（4）セレスナノサイエンス株式会社の間で協力協定。

ヒト血漿および尿がジョージ·メイソン大学施設内倫理委員会は、プロトコールを承認した後に、書面によるインフォームドコンセントを、健康なボランティアドナーから採取した。寄付者は、同様に個別に分析された25および42の試料歳の間白人、男性と女性の間に分布し、プールされませんでした。 血清または血漿検体の1ナノ加工血漿中に潜在的な低豊富なバイオマーカーは、ハイドロゲルナノ粒子で、溶液中で、捕獲されている。粒子は、血漿に添加し培養し、遠心分離によって分離し、洗浄し、捕捉されたタンパク質が溶出されている。溶出したタンパク質は、下流の質量分析配列決定および同定のための窒素気流下で乾燥させる。 <ol><li>マイクロチューブに50mMのトリスHCl pHが7（500μlの血清+500μlのトリスHCl）で2：血清1500μlの希釈する。 </li><li> 500μLのポリ（NIPAM / AAC）コアnanopを追加記事。 RTで15分間インキュベートする。 </li><li>固定角ローターを備えた遠心分離機で10分間、16,100×gで、25℃でサンプルをスピン。上清を除去し、廃棄する。 </li><li>ペレットに500μlのチオシアン酸ナトリウム（25 mm）を追加します。精力的に複数回ピペッティングによりナノ粒子を懸濁します。 </li><li>固定角ローターと遠心機中で10分間、16,100×gで25℃、サンプルをスピン。上清を除去し、廃棄する。 </li><li>ナノ粒子ペレットにMilliQ水を500μlを加える。精力的に複数回ピペッティングによりナノ粒子を懸濁します。 </li><li>固定角ローターと遠心機中で10分間、16,100×gで25℃、サンプルをスピン。上清を除去し、廃棄する。 </li><li>新鮮な溶出バッファーを準備します。清潔なチューブ内の300μlの水酸化アンモニウムを700μlのアセトニトリルを加える。注意：水酸化アンモニウムが腐食性である。適切な換気をすること。注：溶出緩衝液は、予備校でなければなりません使用直前にared。溶出バッファーを保管しないでください。 </li><li>ナノ粒子ペレットに溶出バッファーを300μlを加える。精力的に複数回ピペッティングによりナノ粒子を懸濁します。 RTで15分間インキュベートする。 </li><li>固定角ローターと遠心機中で10分間、16,100×gで25℃、サンプルをスピン。きれいな、ラベルされたマイクロチューブに溶出液を取り出し、保存してください。 </li><li> 1.10 - を繰り返して、1.9を繰り返します。 1マイクロ遠心チューブに2溶出液を組み合わせる。 </li><li> 8（ 図2F）に設定し、空気流で、42.1℃の窒素蒸発器マニホールド内の窒素気流下で溶出液を乾燥させる。 </li><li> O / N個の保存のために室温で乾燥させ、溶出液を保管したり、長期保存のために-20℃、での質量分析、ウェスタンブロッティング、またはELISAアッセイ（オプション）の前に。 </li></ol>尿サンプルの2ナノ加工正常な尿未満では30mg / dlのタンパク質を含有し1 +血液よりも少ない。しかし、多くの疾患/状態は、尿タンパク、血液の正常なレベルを変化させることができる。尿サンプルに追加するナノ粒子の最適な量を決定するのを助けるために、尿検査は、ナノ粒子の収穫に先立って行われる。尿バイオマーカーは、尿量にナノ粒子の比率を最適化する必要があり、極めて低濃度で存在することができる。この手順は、下流のウェスタンブロット分析のための尿サンプルのナノ粒子収穫を記述する。 <ol><li>清潔で乾燥したプラスチック製のカップに尿サンプルを収集します。 22ミリリットル最小体積が必要です。分析する直前まで、-80℃で保存する尿検体。 </li><li> RTで、または4℃CO / Nで凍結した尿を解凍する。ボルテックスミキサーで簡単にミックス。 50ミリリットルコニカルポリプロピレンチューブに尿を少なくとも22ミリリットルを注ぐ。 </li><li> 15分間3,700×gで、スイングアウトローターを遠心分離器で尿をスピン。清潔な50mlコニカルボトムpolyprに、ペレットを乱すことなく、尿をデカントopyleneチューブ。ペレットを捨てる。 </li><li>多検体 &quot;尿ディップスティック」試薬ストリップを使用して尿検査を行います。注意：直射日光を避け、湿度17から密閉容器内で18を試薬ストリップを格納します。 <ol><li>清潔で乾燥したペーパータオル上、試薬ストリップ、フェイスアップにして置きます。 </li><li>ステップ2.3で調製した尿1ミリリットルを吸引する使い捨てピペットを使用してください。 </li><li> 2分間タイマーを設定し、それを起動しないでください。試薬ストリップの各テストパッドの上に尿を2滴 - すばやく1を分注する。すぐにタイマーを開始。注：尿容器内に直接試薬ストリップを水没しないでください。試薬ストリップ中の染料/化学的指標は、潜在的に尿容器に試薬ストリップから浸出することができます。 </li><li>試薬ストリップ容器に記載されている時には、対応する色分けされたインディカに個別の試薬ストリップの色を比較することにより、試薬ストリップ上のさまざまな分析物について定性的な結果を記録試薬容器上の役。典型的な正常な尿の結果は以下のとおりです（正常または負）血液、タンパク質、白​​血球、亜硝酸塩、グルコース、ケトン、ビリルビン、およびウロビリノーゲンのために;尿pH（5.5から7.0）;比重（1.001から1.020）。注：尿試料での高タンパク質レベルは、ナノ粒子上の結合部位について目的のタンパク質と競合しうる。ナノ粒子を有するタンパク質収穫を最大にするために、ナノ粒子の体積/尿の体積比は、高存在量タンパク質のいずれかから限界競争に調整することができる、または非常に低濃度で存在するタンパク質を採取することに最適化することができる。尿タンパクの値が1 +以上の場合、ステップ2.5未満で、ナノ粒子の2倍のボリュームを追加する。目的の分析物が非常に低い豊富なタンパク質である場合、それを2ml（図3）までのナノ粒子の体積を増加させる必要があるかもしれない。 </li></ol></li><li>転送クリーン50ミリリットルコニカルポリプロピレンチューブにステップ2.3から明確に尿を20ミリリットル。しないでください尿管の底部であってもよく、任意の破片/ペレットを乱さ。 20ミリリットルの尿サンプルにナノ粒子を200μlを追加。ボルテックスミキサーで簡単にミックス。尿タンパク値が1+以上の場合、20ミリリットルの尿へのナノ粒子の400μlのを追加してください。 </li><li>ロッキング/ミキシングすることなく、室温で30分間の尿/ナノ粒子混合物をインキュベートする。 </li><li> 10分間3,700×gで、スイングアウトローターを装備した遠心分離機中尿/ナノ粒子懸濁液をスピン。注：ペレットが見え、次の遠心分離がない場合は、追加の2のためのサンプルをスピン - 7分。 </li><li>上清を除去し、廃棄する。ナノ粒子ペレットに500μlのミリQ水を加える。 </li><li>精力的に複数回ピペッティングによりナノ粒子を懸濁します。新しい1.5 mlのマイクロ遠心チューブにナノ粒子溶液を転送します。 </li><li> 10分間16,100×gで固定ローターを装備した遠心分離機でナノ粒子をスピン。注：ペレットが見えFOLされていない場合7分 - 遠心分離をlowing、さらに2のためのサンプルをスピン。 </li><li>上清を除去し、廃棄する。 </li><li>繰り返してナノ粒子を洗浄するために200μlの18ミリQ水（2回）2.8と2.11を繰り返します。 </li><li>新鮮な溶出バッファーを準備します。清潔なチューブ内の30μlの水酸化アンモニウムに970μlのアセトニトリルを加える。注意：水酸化アンモニウムが腐食性である。適切な換気をすること。注：溶出緩衝液は、使用直前に調製しなければならない。溶出バッファーを保管しないでください。 </li><li>ナノ粒子ペレットに20μlの溶出バッファーを追加します。精力的に複数回ピペッティングによりナノ粒子を懸濁します。 RTで15分間インキュベートする。 </li><li> 10分間16,100×gでのナノ粒子のサンプルをスピン。清潔な1.5 mlのマイクロチューブに上清を除去し、保存します。ナノ粒子ペレットを混乱しないでください。バイオハザードゴミ箱にペレットを捨てる。 </li><li>化学ヒュームフード内のラックにサンプルを配置します。キャップを開き、INC室温で30分間ubate。 （ - 2時間1）または、乾燥するまで40℃で窒素流下でサンプルを配置します。 </li><li>サンプルに15μlのトリス - グリシンSDSサンプル緩衝液（2倍）を加える。 100での熱 オープンまたはチューブ内に残って音量まで、キャップを5分間のドライヒートブロック中で°Cを超えない20μlのではない。注：沸騰水浴を使用しないでください。水浴から湿度がバッファの蒸発を防止する。 </li><li>チューブのキャップを置きます。ヒートブロックからチューブを取り外します。サンプルは-80℃で保存するか、または下流のウェスタンブロット分析のために直ちに使用することができる。 </li></ol>

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M., 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=Implication+of+lipocalin-2+and+visfatin+levels+in+patients+with+coronary+heart+disease.">Implication of lipocalin-2 and visfatin levels in patients with coronary heart disease.</a> Eur J Endocrinol. 158 (2), 203-207 (2008).</li><li>Hughes, A. D., Clunn, G. F., Refson, J., Demoliou-Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor+(PDGF):+actions+and+mechanisms+in+vascular+smooth+muscle.">Platelet-derived growth factor (PDGF): actions and mechanisms in vascular smooth muscle.</a> Gen Pharmacol. 27 (7), 1079-1089 (1996).</li><li>Izycki, T., 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=Serum+levels+of+IGF-I+and+IGF-II+in+patients+with+lung+cancer+during+chemotherapy.">Serum levels of IGF-I and IGF-II in patients with lung cancer during chemotherapy.</a> Exp Oncol. 26 (4), 316-319 (2004).</li><li>Jalosinski, M., Karolczak, K., Mazurek, A., Glabinski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+methylprednisolone+and+mitoxantrone+on+CCL5-induced+migration+of+lymphocytes+in+multiple+sclerosis.">The effects of methylprednisolone and mitoxantrone on CCL5-induced migration of lymphocytes in multiple sclerosis.</a> Acta Neurol Scand. 118 (2), 120-125 (2008).</li><li>Kitazume, S., 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=Molecular+insights+into+beta-galactoside+alpha2,6-sialyltransferase+secretion+in+vivo.">Molecular insights into beta-galactoside alpha2,6-sialyltransferase secretion in vivo.</a> Glycobiology. 19 (5), 479-487 (2009).</li><li>Saito, T., 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=Increase+in+hepatic+NKT+cells+in+leukocyte+cell-derived+chemotaxin+2-deficient+mice+contributes+to+severe+concanavalin+A-induced+hepatitis.">Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 2-deficient mice contributes to severe concanavalin A-induced hepatitis.</a> J Immunol. 173 (1), 579-585 (2004).</li><li>Struyf, S., 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=PARC/CCL18+is+a+plasma+CC+chemokine+with+increased+levels+in+childhood+acute+lymphoblastic+leukemia.">PARC/CCL18 is a plasma CC chemokine with increased levels in childhood acute lymphoblastic leukemia.</a> Am J Pathol. 163 (5), 2065-2075 (2003).</li><li>Michael, I. P., 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=Human+tissue+kallikrein+5+is+a+member+of+a+proteolytic+cascade+pathway+involved+in+seminal+clot+liquefaction+and+potentially+in+prostate+cancer+progression.">Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression.</a> J Biol Chem. 281 (18), 12743-12750 (2006).</li></ol>

Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

臨床的関連 血清または血漿試料は、全体としての生物体の状態に関する情報の豊富な供給源を提供することができる低存在量の循環タンパク質およびペプチドを含むと考えられる。血清プロテオミクスの約束にもかかわらず、臨床的有益性10、11、16、25にバイオマーカーの発見および翻訳を阻止3つの基本的かつ重大な生理学的障壁があります。 <...

ヒトゲノムシークエンシングの完了にもかかわらず、かなりの進歩が早期の疾患の予測バイオマーカーの同定に行われていない、またはそれは、治療転帰または予後1と相関する。進歩の欠如の一つの理由は、多くの潜在的に重要なバイオマーカーが従来の質量分析法および他のバイオマーカー発見プラットフォームの検出限界未満の濃度で存在することがある。質量分析（MS）および多重反応モニタリング（MRM）は、分析物の大部分は50 pg / mlで、および10ng / mlの間の範囲の臨床検査秋にイムノアッセイによって測定しながら、ng / mlで50より典型的には検出感度を持つ。これは、特に疾患の早期段階における多くのバイオマーカーは、従来のMS及びMRM 2により検出できないことを意味する。さらに、このような複雑な生物学的流体中のアルブミンおよび免疫グロブリンのような高量のタ​​ンパク質の存在は、しばしば10億倍販売によってマスクエス低存在量、低分子量タンパク質およびペプチド3、図4に示すように 、このためいくつかのサンプル準備ステップは、質量分析配列決定および同定の前に必要とされる。そのような準備工程は、市販の空乏列5-8の高存在量タンパク質の枯渇を採用しています。それらはしばしば、非共有結合的に削除されるキャリアタンパク質と関連しているので、残念ながら、このステップは、候補バイオマーカーの歩留まりの低下につながる。サンプルが収集されると別の課題は、候補バイオマーカーex vivoでの安定性により表される。タンパク質は、内因性または外因性のプロテアーゼ9による分解を受けやすい。分解10-13からタンパク質を保護しながら、ヒドロゲルのナノ粒子は、アッセイの範囲内のレベルまでの推定バイオマーカーの濃度を増幅することにより、これらの重要な課題を超越することができる。 それは、目に注意することが重要ですLMWで血液中のタンパク質は、小さな完全なタンパク質だけでなく、大規模なタンパク質の断片の混合物である。 60kDaのより大きな組織由来のタンパク質は、受動的に、血管基底膜を通って血流に入るには大きすぎるが、それらはペプチドまたはタンパク質断片14として血液中に表すことができる。私たちの目標は、疾病の早期発見、治療のために患者の層別化、および治療に対する応答をモニターするための候補となり得る新規の循環バイオマーカーを測定することである。当社のナノ粒子は、同時により小さなタンパク質およびペプチドを捕捉し、出発容量に応じて、100倍にそれらを集中しながら、選択的に、高存在量の免疫グロブリンとアルブミンを除外するために作成されます。 当社グループは、成功し、タンパク質およびペプチドのための高親和性高分子の餌として作用することができる小さな有機色素のセットを同定した。タンパク質 - 色素結合、疎水性及び静電的相互作用の組合せに起因すると考えられているS。タンパク質表面11上の疎水性ポケットを経由してタンパク質と色素のインターリーブ上の芳香環。 餌は、それらの化学的性質に応じて、分析物の選択されたクラスに対して特定の親和性を示す。餌は、タンパク質またはペプチドを、アルブミンなどのキャリアタンパク質と競合する。粒子中に捕捉される低分子量タンパク質/ペプチド。例えば、アルブミンおよび免疫グロブリンなどの高分子量タンパク質に起因ヒドロゲル11（図1）の制限のため、細孔にふるい分け能力の粒子に侵入することを防止している。 ヒドロゲルのナノ粒子は、過硫酸アンモニウム11によって開始沈殿重合により合成される。 N-イソプロピルアクリルアミド（NIPAM）は、アクリル酸（AAC）およびアリルアミン（AA）及び架橋剤N、N&#39;-メチレンビスアクリルアミド（BIS）のコモノマー、希釈条件1を 6時間70℃で反応させ1,13。共有結合を介してナノ粒子にアミノ基含有染料（ すなわち 、sulfonatedanthraquinonetriazine染料）を組み込むことによって達成ポリ親和性（N-イソプロピルアクリルアミド-コ-アクリル酸）（ポリ（NIPAM-コ-AAC）nanoparticlesis結合高タンパク質染料11、13の親水性/疎水性特性に依存して水性または有機溶媒中で実施アミド化反応。anthraquinonetriazine色素の塩素原子を有するナノ粒子中のアミン基の求核置換は、色素含有ポリ（NIPAMを作成するために利用されるコ-アリルアミン）（AA）は、11ナノ12。二段階の重合プロセスは、ビニルスルホン酸（VSA）11,13の外殻を含むヒドロゲルのナノ粒子を作成するために利用される。 ヒドロゲル粒子は、全血、血漿、血清、脳脊髄液、汗、および尿を含むさまざまな生物学的液体に適用することができる。ワンステップで、溶液中で、nはanoparticlesは（数分以内）急速隔離および低分子量の濃度は10、11、13、15-18分析物行う。タンパク質は、続いて19-21、質量分析法10、11、13、15、18、22、23、イムノアッセイ/ ELISA 10、11、15、18、または逆相タンパク質マイクロアレイ16を、ウェスタンブロッティング用いてナノ粒子から溶出し、検出され24アッセイ。化学餌で官能ナノ粒子と、コアまたはコアシェル·アーキテクチャを提示するには、餌/シェルの物理化学的特性に基づいてタンパク質を捕捉し、濃縮する。ナノ粒子に組み込まれた別の染料は、したがって、色素親和性に基づいて効率、溶液のpH、高濃度タンパク質13、競合の存在/不在を変化させて、タンパク質の異なるサブセットを捕捉するであろう。さらに、溶液の体積に対するナノ粒子の量は、ナノ粒子からのタンパク質収量に影響する。これらの態様のハイドロゲルナノ粒子収穫は、タンパク質を大量に含有して血漿サンプルから、典型的には大量のタンパク質を含まない尿サンプルからタンパク質を回収するための3つの異なるナノ粒子ベイトを用いて実証されている。このプロトコルでは、収穫およびポリ（NIPAM-コ-AAC）、ポリ（NIPAM /染料）、及びコア - シェル型ナノ粒子を用いて血漿サンプルからの腫瘍壊死因子α（TNFα）の濃縮を実証する（ポリ（NIPAM - コ - VSA）） 。ポリ（NIPAM /色素）のナノ粒子は、 結核菌感染個体を模倣するために、ヒト尿サンプルに添加したマイコバクテリウム種の抗原を濃縮することが示されている。

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">hydrogel nanoparticles</td>
			<td style="width:187px;">Ceres Nanoscience</td>
			<td style="width:119px;">CS003</td>
			<td style="width:336px;">NanoTrap ESP particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl, 50mM pH7.0</td>
			<td style="width:187px;">VWR</td>
			<td style="width:119px;">IC816116</td>
			<td>50mM, pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH1103-4LP</td>
			<td>available from VWR</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium Hydroxide NH4OH</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH3014</td>
			<td>available from VWR, assayed at 28-30% NH3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiocyanate 25mM</td>
			<td style="width:187px;">Acros Organics</td>
			<td style="width:119px;">419675000</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-analyte Urine Reagent Strips</td>
			<td style="width:187px;">Siemens</td>
			<td style="width:119px;">2161</td>
			<td style="width:336px;">for urine samples</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tris-Glycine SDS Sample Buffer (2X)</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">LC2676</td>
			<td style="width:336px;">use at room temperature to prevent SDS from precipitating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dry bath incubator (100 oC) with heating block</td>
			<td style="width:187px;">Barnstead</td>
			<td>11-715-125DQ</td>
			<td>do not substitute a boiling water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator manifold</td>
			<td style="width:187px;">Organomation Associates</td>
			<td>Microvap118</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, swing-out rotor</td>
			<td style="width:187px;">Sorvall</td>
			<td style="width:119px;">Legend series</td>
			<td>50ml tube capacity, rcf 3700 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, fixed angle rotor</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;">5424</td>
			<td>1.7ml microcentrifuge capacity, rcf 16,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50ml conical centrifuge&#160;tubes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td>with screw caps for urine samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1.5ml microcentrifuge tubes</td>
			<td style="width:187px;">Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Disposable plastic transfer pipettes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7M</td>
			<td>at least 1ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>50-949-755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Timer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">S04782</td>
			<td>seconds/minutes</td>
		</tr>
	</tbody>
</table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

新規バイオマーカーの発見は、より高感度かつ特異的な疾患検出を提供する上で重要な役割を果たしている。それらは、非常に低濃度で存在する不安定であり、しばしば、アルブミンまたは免疫グロブリンのような高量のタ​​ンパク質によってマスクされるため、残念ながら、生物学的流体中に存在する多くの低存在量のバイオマーカーは、容易に、質量分析またはイムノアッセイを用いて検出することができない。ポリ（N-イソプロピルアクリルアミド）（NIPAM）ベースのナノ粒子を含有する餌は、これらの生理学的障壁を克服することができる。一工程において、それらは、捕捉し濃縮して、体液からのバイオマーカーを保持することができる。低分子量の分析物は、ナノ粒子のコアに入り、高親和性タンパク質ベイトとして作用する異なる有機化学色素によって捕捉される。ナノ粒子は、数桁目的のタンパク質を集中することができます。この濃縮係数は、タンパク質が範囲内にあるように、タンパク質レベルを増加させるのに十分である現在の質量分析計、ウェスタンブロッティング、および免疫アッセイの検出限界。ナノ粒子は、生物学的流体の過多とインキュベートすることができ、それらは、アルブミンおよび他の高分子量蛋白質を除外して大幅に低分子量タンパク質およびペプチドの濃度を豊かにすることができる。本発明者らのデータは、以前に検出できないバイオマーカーを検出するために質量分析および免疫アッセイを可能にする、特定の分析物の濃度の10,000倍の増幅が達成できることを示している。

ハイドロゲルナノ粒子サイズと均一性 ポリ（NIPAM-AAC）粒子間のバッチ内で極めて高い収率や再現性で製造されてきた。粒子が捕捉、貯蔵、およびタンパク質の溶出（少なくとも48時間）に必要な時間の間、室温で非常に良好なコロイド安定性を有し、ナノ粒子沈殿（ 図1）11 を観察されていない。コロイド安定性は、ナノ粒子に?...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

ハイドロゲルナノ粒子プラズマの収穫や少量のタンパク質を検出するための尿

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

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13.8K ビュー.  George Mason University. Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

ビデオ: Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

細胞ネットワークの作成のためのプラズマのリソグラフィ表面パターニング

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

生物工学

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3次元自己組織化ペプチドハイドロゲル中のヒト神経前駆細胞の栽培

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

人工細胞外マトリックス（AECM）タンパク質からの設計と構築<em&gt;エシェリヒア·コリ</em&gt;皮膚組織工学のための

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

繊細な組織操作のためのカスタマイズ可能なソフトロボット空気圧グリッパ装置のロッドベースの試作

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

液晶ナノ粒子キャリアにおける疎水性貨物カプセル化の標的細胞膜配達

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

配向柱状組織を採取するための装置

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

細菌スクリーニング、選択、および分離のための光分解性ヒドロゲルインターフェース

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

強接着性ハイドロゲルゼラチンO-ニトロソベンズアルデヒドの合成

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...