我々は、基礎となる研究の部分的なサポートについては、この原稿のサポートのためにとONR、DTRA、およびNSFからDTRAからの資金調達を認める。

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+"dual+mode"+actuation+in+chitosan/polyaniline/carbon+nanotube+fibers.">A novel "dual mode" actuation in chitosan/polyaniline/carbon nanotube fibers.</a> Sensor Actuat B-Chem. 121, 616-621 (2007).</li><li>Yi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+assembly+of+genetically+modified+viral+nanotemplates+via+nucleic+acid+hybridization.">Patterned assembly of genetically modified viral nanotemplates via nucleic acid hybridization.</a> Nano letters. 5, 1931-1936 (2005).</li><li>Kim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox-cycling+and+H2O2+generation+by+fabricated+catecholic+films+in+the+absence+of+enzymes.">Redox-cycling and H2O2 generation by fabricated catecholic films in the absence of enzymes.</a> Biomacromolecules. 12, 880-888 (2011).</li><li>Xie, Y., Xu, B., Gao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+transdermal+delivery+of+model+drug+compounds+by+MEMS+microneedle+array.">Controlled transdermal delivery of model drug compounds by MEMS microneedle array.</a> Nanomedicine. 1, 184-190 (2005).</li><li>Odaci, D., Timur, S., Telefoncu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microbial+biosensor+based+on+bacterial+cells+immobilized+on+chitosan+matrix.">A microbial biosensor based on bacterial cells immobilized on chitosan matrix.</a> Bioelectrochemistry. 75, 77-82 (2009).</li><li>Selimoglu, S. M., Elibol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+as+an+immobilization+material+for+MAb+production+via+encapsulated+hybridoma+cells.">Alginate as an immobilization material for MAb production via encapsulated hybridoma cells.</a> Crit Rev Biotechnol. 30, 145-159 (2010).</li><li>Braschler, T., Johann, R., Heule, M., Metref, L., Renaud, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentle+cell+trapping+and+release+on+a+microfluidic+chip+by+in+situ+alginate+hydrogel+formation.">Gentle cell trapping and release on a microfluidic chip by in situ alginate hydrogel formation.</a> Lab Chip. 5, 553-559 (2005).</li><li>Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-Film+Bioprocessing+and+Immunoanalysis+with+Electroaddressable+Stimuli-Responsive+Polysaccharides.">In-Film Bioprocessing and Immunoanalysis with Electroaddressable Stimuli-Responsive Polysaccharides.</a> Adv. 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この記事への生産とフリーアクセスは、米国の国防脅威削減局が主催しています。

私たちの手順では、高分子膜の電着と官能、biofabrication我々は長期的なプロセスを示しています。細胞や生体分子の機能化を通して、私たちはお互いに、それらが上に組み立てられる電極のアドレスと相互作用することができる生物学的な表面を作成します。最初のステップは、電着は、電気信号​​に応答して、我々の研究では生体高分子、アルギン酸塩、キトサンのトリガ自己組織化を介して行われます。述べたように、以前のpH勾配は6,17フィルムの寸法と特性を詳細に制御を提供し、電流密度と成膜時間で制御することができる生成されます。我々は電流密度の多様性と堆積時間の組み合わせは表1に示した電極に使用することができることを発見した。他の電極の使用は可能ですが、プロシージャへの調整が必要であろう。膜形成の他の技術electrodeposiのプロセスと比較してるには、単純、迅速かつ反応試薬である。高価な装置と骨の折れる準備の豊富なレパートリーの必要はありません。重要なのは、プロセスでは、マイナーな実験的な偏差を耐えることができ、問題が発生した場合、簡単に上を開始することができます。 キトサンは、第一級アミンの含有量が高いことによって、それに付与される重要な機能的特性に起因する高い陰極のpH勾配に応答することができます。高いpH（〜6.3のそのpKaがより大きい）で、アミンが脱プロトン化とキトサンは膜形成が可能になり、不溶化されています。堆積後、フィルムが電極に接続されたままになります。しかし、能力は必要に応じてそれらを剥離するために存在します。フィルムは、溶液のpHがpKa値を下回らない限り、安定したままになります。酸性溶液は、アミンをプロトン化し、それが18を溶解するまで、後続の静電反発は、ゲルを膨潤させる。つまり、組立/分解プロセスでは、需要と同種で可逆的である蒸着膜と電極の再利用を除去するためのWS。好都合なことに、ゾル - ゲル転移が行われるのpHの範囲は、ほとんどの生物学的なコンポーネントが最適に機能しているに近いです。これは、アセンブリ6時の機能の保持のためのプロセスが理想的です。 アルギン酸膜の形成は、水の陽極電解と同様に炭酸カルシウム7の存在によって促進される。アノードではローカライズされた低pHは、リリースカルシウムカチオンにつながる炭酸カルシウムを溶解。これらのイオンは電極表面上に架橋ネットワークを形成し、アルギン酸塩でキレートされています。アルギン酸塩フィルムは、基礎となる電極の再利用を可能にし、フィルムを溶解するために使用することができるようにクエン酸やEDTAなどの他のキレート化合物からのカルシウムイオンの競合により、特に可逆的である。カルシウムイオンが容易に秒であるため、このように、アルギン酸塩フィルムは、生理学的条件にさらされたとき比較的脆いその構造を弱体化、フィルム剥離や再溶解を促進し、ゲルマトリックスからcavenged。この制限を克服するために、我々はゲルを強化するため、1 M CaCl 2でのフィルムのためのインキュベーションステップが含まれている。さらに、我々は映画の培養液（細胞培地など）は、500μM-3 mMの濃度でCaCl 2を補足することをお勧めします。 番目の主要な手順は、関連する生物学的成分と堆積膜の機能化である。これには二つの方法は、最初である電気化学的接合、卓越した空間的制御10のタンパク質の迅速、反応試薬アセンブリを可能にする戦略で達成することができます。しかし、このように官能は、Clの拡散によって制限されます-電極に膜を介してイオンだけでなく、HOClの拡散、溶液中に戻って生成された反応性中間体、アウト。渡すための電気化学的に活性な分子の能力膜を介して読みやすい電気信号15に化学的および生物学的シグナルの伝達を可能にします。我々は、共有AI-2酵素を取り付けることによって実証し、キトサンの酵素機能化のために第二の戦略​​としてチロシナーゼを介したカップリングを示している。チロシンタグ9を含む蛋白質で差別的役割を果たし、特定の試薬 ​​、チロシナーゼに依存-この戦略は、官能化プロセスが制御され、選択にすることができます。 我々は、チップ上に自然な経路を複製することによって、マルチアドレスシステムの有用性と生体適合性を示しています。まず、異なるアドレスで2つの細胞集団（すなわち、 &quot;送信者&quot;と &quot;受信機&quot;）を組織し、彼らはAI-2を提供し、蛍光応答を生成するために、隣接する電極間に相互作用することを示した。この概念はまた、チェンらによって実証されています。マイクロ流体チップ14インチまた、相互作用を模倣するのではなく、使用配信のためにAI-2を合成する酵素。このように、合成細胞内経路、AI-2の合成に、biofabrication介してレプリケートして、溶液中と同じように非常に機能した。 両方のケースでは、複数のアドレスのアセンブリは、それぞれの堆積溶液が電着が1つだけのアドレスで意図されているにもかかわらず、全体の電極アレイを導入する必要があるため、アドレスの間の非特異的結合を回避するための課題となっています。穏やかな、まだ徹底した洗浄では、非バイアス電極からの残液の大部分を削除することができます。マイクロチャネル内の流れの使用は、さらに非特異性を最小限に抑えることができます。特にキトサンとアルギン酸アドレスの隣接biofabricationために、我々は、アルギン酸塩を電着、最初のキトサン膜を堆積biofunctionalizationの手順でこれを後にし、この後をお勧めします。私たちはそうここに行っていないが、我々は、ミルのような不活性蛋白質（キトサンフィルムを阻止することを見出したK、BSAなど）が大幅にキトサンのアミノ表面に不要な分子の非特異的結合を減少させる。 我々は、細胞や生体分子の複雑な配置のための &quot;青写真&quot;として、多くの場合、バイオMEMSデバイスに見られるパターン化電極を、確立するユーティリティを発見した。バイオMEMSデバイスにキトサンを電着の用途はよく19ここで説明した例を越えて行くことができます。このようなマイクロおよび非平面20,15上と-キトサンは、様々なマイクロジオメトリ上に堆積することができます。フィルムはまた、他のポリマーとの様々な蛋白質、DNA、ナノ粒子、および新規のプロパティ21,22,23ためのレドックス活性分子を変更することができます。バイオMEMSデバイスでは、キトサンフィルムは、薬物送達、レドックスおよび小分子の検出、生体触媒、およびセルの研究20,23,24,25に使用されています。同様に、アルギン酸塩は、広く細胞封入マトリックスとして使用されているとの可逆的流体封じ込めのために検討されている細胞集団との膜免疫分析26,27,28。組織工学アプリケーション用の複合フィルムは、そのような整形外科用インプラント29ハイドロキシアパタイトと同様に、コンポーネントを使用して、アルギン酸電を用いて作製されています。 biofabricationの我々のデモでは、生体成分の間に、バイオ、電子インタフェースを介して相互作用の両方が等しく適用可能であることが示されているが、これはオンチップの信号伝送で洗練されたパフォーマンスを得るために相互作用のすべての品種を統合するための見通しを達するにもたらします。したがって、biofabricationフォローオンとしてしばしばコンシューマエレクトロニクスによって動機づけられて微細加工の急速な発展に直接に低減 &quot;最小特徴サイズ&quot;でデバイスの製造を容易にすることができる。つまり、次の次世代デバイスは、実際にはさらに小さい長さSCAにおける自然の絶妙な組み立ておよび認識機能を提供する不安定な生体成分が含まれる場合があります人工システムに比べてレ。我々は、分析機器、環境センサー、さらには生体適合性植込み型デバイスの短期的なアプリケーションを想定。

<table border="1"><tbody><tr><td> コンポーネントの名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>パワーサプライ</td><td>ケースレー</td><td>ソースメータ2400 </td></tr><tr><td> 3電極ポテンショスタット</td><td> CHインスツルメンツ</td><td>ポテンシオスタット/ガルバノスタット600D </td></tr><tr><td>フレキシブルコネクタ付きRE-5B Ag / AgCl参照電極</td><td>バシ</td><td> MF-2052 </td></tr><tr><td>ゴールドコーティングされたシリコンウェハ、500umのSi、12nmのCrを、120nMのAu、絶縁用のSiO 2 </td><td>カスタム製作</td><td></td></tr><tr><td>インジウムスズ酸化物被覆ガラススライド、長方形、8から12オーム抵抗する</td><td> Sigma-Aldrich社</td><td> 578274 </td></tr><tr><td>プラチナシート/箔（0.002） </td><td> SurepureのChemetals </td><td> 1897 </td></tr><tr><td>スリムライン2 &quot;アリゲーター·クリップ</td><td>ラジオシャック</td><td> 270から346 </td></tr> &lt;TR&gt; <td>マルチスタッキングバナナプラグパッチコード</td><td> TSElectronic </td><td> B-36から02 B-24から02 </td></tr><tr><td> SYLGARD 184シリコーンエラストマーキット</td><td>ダウコーニング</td><td> NC9020938 フィッシャーから</td></tr><tr><td> Fluorescecenceの実体顕微鏡</td><td>オリンポス</td><td> MVX10 MacroView </td></tr><tr><td> cellSens標準</td><td>オリンポス</td><td>バージョン1.3 </td></tr></tbody></table> 表1電と蛍光可視化装置。 <table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>キトサン、中分子量</td><td> Sigma-Aldrich社</td><td> 448877 </td></tr><tr><td>塩酸、ARISTAR。 ACS、NF、FCCグレード</td><td> VWR </td><td> BDH3030 </td></tr><tr><td>水酸化ナトリウム、ソリューション。 10.00N </td><td>VWR </td><td> VW3247 </td></tr><tr><td>アルギン酸、ナトリウム塩</td><td> Sigma-Aldrich社</td><td> 180947 </td></tr><tr><td> Multifex-MMは、沈殿 炭酸カルシウム、70nmの粒子</td><td>特殊 ミネラル株式会社</td><td> 100-3630-3 </td></tr></tbody></table> 表2。キトサンとアルギン酸塩溶液試薬。 <table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>塩化カルシウム二水和物</td><td> JTベーカー</td><td> 0504 </td></tr><tr><td>塩化ナトリウム、認定 結晶ACS </td><td>フィッシャー 科学的な</td><td> S271 </td></tr><tr><td>リン酸二水素カリウム、無水</td><td> Sigma-Aldrich社</td><td> P9791 </td></tr><tr><td>リン酸カリウム二塩基、無水</td><td> Sigma-Aldrich社</td><td> P3786 </td></tr><tr><td>リン酸緩衝生理食塩水</td><td>シグマ アルドリッチ</td><td> P4417 </td></tr></tbody></table> 表3その他のソリューションコンポーネントとバッファ試薬。 <table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社/ソース </td><td> カタログ番号 </td></tr><tr><td> アスペルギルスニジェールからのグルコースオキシダーゼ</td><td> Sigma-Aldrich社</td><td> G2133 </td></tr><tr><td>キノコからのチロシナーゼ</td><td> Sigma-Aldrich社</td><td> T3824 </td></tr><tr><td> LBブロス、ミラー（粒状） </td><td>フィッシャーサイエンティフィック</td><td> BP9723-2 </td></tr><tr><td> &quot;AI2-シンターゼ&quot;（HGLPT） </td><td>ラボ株式16 </td><td></td></tr><tr><td> W3110野生型細胞</td><td>ラボ株式30 </td><td></td></tr><tr><td> MDAI2 + pCT6-LSRR - AMP R + PET- <eM&gt; DsRedを- 館 r細胞</td><td>ラボ株式30 </td><td></td></tr><tr><td>蛍光粒子：1μmの直径、EX / EM：515分の505 </td><td>インビトロジェン</td><td> F8765 </td></tr><tr><td> 5 - （および6） - カルボキシ6Gスクシンイミジルエステル、EX / EM：560分の525 </td><td>インビトロジェン</td><td> C-6157 </td></tr><tr><td> DyLight抗体ラベリングキット、405 </td><td>サーモフィッシャーサイエンティフィック</td><td> PI-53020 </td></tr></tbody></table> 表4。酵素、細胞、および他の官能試薬。

bridging the bio-electronic interface with biofabrication

低コスト、優れた感度、移植性、および高いスループットによる研究と医学の両方を変革するためのラボオンチップ技術の約束の進歩。生物学的微小電気機械システム（バイオMEMS）の上に生体成分の混入は、これらの目標を達成するための大きな可能性を示している。微細加工、電子チップは、マイクロメートルスケールでの機能に加えて、センシングおよびアクチュエーションのための電気的接続を可能にします。機能的な生物学的なコンポーネントは、システムの測定対象化合物、酵素の機能、および全細胞機能の特異的検出のための能力を与える。標準的な微細加工プロセスとバイオ分析技術が正常に、それぞれのコンピュータと生物学的な産業における数十年にわたって利用されている。ラボオンチップ環境でそれらの組み合わせとのインタフェースは、しかし、など新たな課題をもたらします。電極と生物学的compon間のインタフェースを構築できる技術のための呼び出しがあります穏やかで、作製したパターンが容易であるENT。 Biofabricationは、ここで説明され、有効になっているオンチップ機能に汎用性を備えた生体成分のその、簡単に組み立てるの取り込みのために偉大な約束を示しているそのようなアプローチである。 Biofabricationは、ボトムアップの階層的なアセンブリに生物学的物質および生物学的メカニズム（自己組織化、酵素のアセンブリ）を使用します。私たちのラボは多くのフォーマット1,2,3にこれらの概念を実証しているが、ここでは、シグナル·ベースの相互作用の複数のアプリケーションに続いて電着に基づいて、組立工程を示しています。組立工程は、電極及びそのようなDNA、酵素、または4,5 -生きている細胞などの生体成分とのその後の官能基に生体適合性の刺激応答性高分子膜の電着で構成されています。電着は、電解からバイアス電極の表面で作成されたpH勾配を利用しています、水6,7の。キトサンおよびアルギン酸塩は、8課した電気信号に応答して、ハイドロゲルフィルムに自己集合するようにトリガーすることができる刺激応答性の生物学的ポリマーである。これらのハイドロゲルの厚さは、pH勾配が電極から延びるする程度によって決定されます。これは、様々な電流密度と堆積時間6,7を使用して変更できます。このプロトコルは、キトサンフィルムは共有結合酵素的または電気化学的方法9,10のいずれかを介してフィルム上に存在する豊富な第一級アミン基に生体成分を付加することによって堆積し、官能化され方法について説明します。アルギン酸塩フィルムと生細胞のそれらの捕捉も11を対処されることになります。最後に、biofabricationのユーティリティは、化学 - 電気、細胞から細胞へ、また酵素の細胞信号伝送を含む信号ベースの相互作用の例を通じて実証されています。 両方の電と官能基は、試薬を必要とせずに近い生理的条件下で行うため、過酷な条件から不安定な生体成分を割くことができます。さらに、キトサン、アルギン酸塩の両方が長い生物学的に関連の目的で12,13のために使用されている。全体として、biofabrication、単にベンチ上で実行することができます急速な技術は、電極上に機能的な生体成分のミクロンスケールのパターンを作成するために使用することができ、ラボ·オン·チップのさまざまなアプリケーションに使用することができます。

Watch this Scientific Journal Video about Bridging the Bio-Electronic Interface with Biofabrication at JoVE.com

Bridging the Bio-Electronic Interface with Biofabrication

細胞やタンパク質と機能化することができる生体適合性フィルムを作成するためのバイアス電極の存在下で刺激応答性多糖類の沈着：この資料では、biofabricationアプローチを説明しています。我々は、ラボ·オン·チップ·アプリケーション用の対話biofunctionalizedサーフェスを作成するためのフィルムだけでなく、その基本的な用途を生成するためのベンチトップ型の戦略を示しています。

Biofabricationとバイオエレクトロニック·インタフェースをブリッジする

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

bridging-the-bio-electronic-interface-with-biofabrication

Research

JoVE Journal

Bioengineering

16.7K ビュー.  University of Maryland. 細胞やタンパク質と機能化することができる生体適合性フィルムを作成するためのバイアス電極の存在下で刺激応答性多糖類の沈着：この資料では、biofabricationアプローチを説明しています。我々は、ラボ·オン·チップ·アプリケーション用の対話biofunctionalizedサーフェスを作成するためのフィルムだけでなく、その基本的な用途を生成するためのベンチ?...

ビデオ: Bridging the Bio-Electronic Interface with Biofabrication

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

用接着周波数アッセイその場で</em細胞間のインタフェースにおけるクロス接合分子間相互作用の>速度論解析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

生物工学

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

シルバーシード媒介成長法によるゴールドNanostarの合成

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

蛍光顕微鏡を用いたイメージング細胞遊走用接着剤及び可溶性グラデーションを作成する

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

用アメロゲニン - キトサンハイドロゲルの開発<em&gt;体外</em高密度インターフェイス付き&gt;エナメル再成長

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

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

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

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

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

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

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

プログラム可能な材料とのインタフェースリビング細胞を操作するために合成生物学を使用して、

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

成体ラットにおける複合再生性末梢神経インタフェース(C-RPNI)の作製

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

超高速ドップラーによる血流イメージング

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

液滴界面二層の温度制御アセンブリと特性

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

透過処理検出機能付き連続フローマイクロエレクトロポレーションシステムの構築と運用

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...