Name: advanced compositional analysis of nanoparticle-polymer composites using direct fluorescence imaging
Uploaded: 2016-07-19T14:00:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging at JoVE.com

C.R.C. would like to acknowledge the Ramsay Memorial Trust for funding.

CdSe / ZnSのコア/シェル量子ドットの作製<ol><li> トリオクチルホスフィンの調製（TOP）-Seソリューション <ol><li>窒素下またはグローブボックス中のシュレンクフラスコにTOPへのSeの適切な量を混合することにより、TOP中のセレンの0.5M溶液（反応ごとに必要な8ミリリットル、TOPの10ミリリットルに溶かし、通常、0.4グラム）を準備します。 </li><li> TOP-Seの複合体の灰色溶液が得られ、1時間のSeを溶解するために混合物を撹拌しました。 </li><li>溶液を凍結 - ポンプ - 解凍を5回脱気した後であることを確認します。得られたストック溶液は、3ヶ月間、窒素下で保存することができます。 </li></ol></li><li> CdSeコアの調製 <ol><li>酸化カドミウム（51ミリグラム、0.4ミリモル）、TOP酸化物（3.7グラム、9.6ミリモル）、ヘキサデシルアミン（1.93グラム、8ミリモル）及び1-ドデシル酸（0.22グラム、0.88ミリモル）を秤量し、3首に結合し、250 mlの丸底フラスコ。攪拌棒を追加します。 </li><li>隔壁を2首を閉じ、確保第三は、長い還流冷却器および窒素/真空シュレンクラインに取り付けられています。混合物に直接1セプタムを通して加熱マントルの温度プローブを挿入します。ポンプ/窒素で5回フラスコを補充します。 </li><li> 320℃までフラスコを加熱し、窒素雰囲気下で1時間溶融物を攪拌します。 </li><li> 270°Cまでのマントルピースの温度を下げた後、窒素で脱気に5回大きな注射器と広い口径の針（20ミリリットル、3ミリメートルの穴）を使用します。 </li><li>アップTOP-Seの溶液（ステップ1.1）の8ミリリットルを取り、セプタムを通して、3口フラスコに慎重が、急速に注入します。 </li><li> secで製造された粒子のサイズを制御するために、10分間、30〜270℃で反応を撹拌しました。赤色発光（〜600 nm）のために、7~9分が好適です。 </li><li> （反応容器の半分水没するのに十分な大きさ）を加え、反応混合物を次に沸騰したお湯を入れたボウルを準備します。反応時間が完了した後、急速にboili反応を冷却かき混ぜながら水をngの。 注意：冷却はフラスコに亀裂を引き起こす可能性があります。細心の注意を払って、厚い不浸透性の手袋を着用してください。 </li><li>冷却したら、すべての製品を溶解し、2つの50ml遠心管の間の混合物を分割するためにフラスコに10mlのクロロホルムを注入。 </li><li> 3600×gで10分で各チューブアップエタノール、遠心分離で50mlにトップアップは、粒子を沈殿させました。上清を捨て、n-ヘキサン10ミリリットルの合計でペレットを再分散させます。 </li></ol></li><li> CdSeコアのZnSの砲撃： <ol><li> 100mlにヘキサン中のコアを追加ジエチルジチオカルバミン酸亜鉛（0.8 gであり、1.4ミリモル）を含有する丸底フラスコに、オレイルアミン（3 mlの9.12ミリモル）、トリオクチルホスフィン（3 mlの6.73ミリモル）及び1-オクタデセン（10ml）溶液。攪拌棒を追加します。窒素に反応雰囲気を交換します。 </li><li> 70°Cまで、部分真空下で3.3℃/分でホットプレート攪拌機上の反応を熱し、そしてシュレンクラインを使用してヘキサンを除去。 </li&gt; <li>窒素に雰囲気を切り替えて、120℃にこのレートで加熱を続けます。 2時間120℃で撹拌します。 </li><li>反応を冷却することを可能にし、2×50mlの遠心分離管の間の混合物を分割します。チューブを10分間3600×gで粒子と遠心分離を沈殿させEtOHで50ミリリットルまで突破していることを確認します。 </li><li>上清を捨て、n-ヘキサン10ミリリットルの合計でペレットを再分散させます。 </li><li>遠心分離機この溶​​液（3600×gで、10分）を1回以上までの3ヶ月間、サンプル管にデカントされる前に、任意の不溶性不純物を除去し、窒素雰囲気下、冷蔵庫（4℃）に格納します。 </li></ol></li></ol> PDMSへのナノ粒子の2膨潤カプセル化<ol><li> 溶液の調製を膨潤： <ol><li> 4（合成される）のCdSe量子ドットの分散液mlのn-ヘキサン36ミリリットルを混合したCdSe量子ドットのストック溶液を調製し、磁気的に溶液を攪拌します。&lt;/李&gt; <li>別に指定腫れソリューションとして原液の9ミリリットルを含む2つのバイアルをそれぞれ設定します。 </li><li> QD濃度を変化させ、さらに膨潤溶液の調製のためのストック溶液の残りの部分を使用してください。 66％（v / v）の溶液を、50％溶液および33％の溶液を得たストック溶液を希釈することによって減少QD濃度の三膨潤溶液を調製します。 <ol><li> n-ヘキサン3mlでのCdSe QDストック溶液を6mlを混合することにより、66％（v / v）の溶液を調製します。完全な混合を確実にするために、磁気的に溶液を攪拌します。 </li><li> 4.5 mlのn-ヘキサンでのCdSe QDストック溶液4.5mlを混合することによって、50％（v / v）の溶液を調製します。完全な混合を確実にするために、磁気的に溶液を攪拌します。 </li><li> n-ヘキサン6ミリリットルでのCdSe QDストック溶液3mlを混合することにより、33％（v / v）の溶液を調製します。完全な混合を確実にするために、磁気的に溶液を攪拌します。 </li></ol></li><li>室温で、暗条件下でQDソリューションのすべてを格納します。&lt;/李&gt; </ol></li><li> ポリマーの調製およびQD取り込み-膨潤液にQDの濃度を変化させること： <ol><li>新鮮な手術用メスの刃を使用して、4つの医療グレードのシリコーンの正方形（11ミリメートル×11 mm）を切り出します。 </li><li>変化％のQD濃度の4膨潤溶液のそれぞれにおける医療グレードシリコーン正方形浸し：ストック溶液を、66％（v / v）で、50％（v / v）の33％（v / v）です。ポリマーサンプルは、暗条件下で、及び室温で24時間膨潤することを可能にします。 </li><li>残留溶剤が蒸発し、ポリマーが戻って自分の初期の寸法に縮小された時間の間に、48時間暗条件下でそれぞれの腫れソリューション、空気乾燥から膨潤したポリマーサンプルを削除します。 </li><li>任意の表面に結合した物質を除去するために脱イオン水で十分にQD-組み込まれたサンプルを洗ってください。 </li></ol></li><li> ポリマーの調製およびQD取り込み-膨潤液にポリマーの露出時間を変えます： </stronグラム&gt; <ol><li> 2.2.1で述べたように、4つの医療グレードのシリコーンの正方形（11ミリメートル×11 mm）を準備します。 </li><li>期間を変化させるための解決策を膨れ入荷医療グレードのシリコーンの正方形を浸し：1時間、3時間、6時間および24時間。 </li><li>膨潤液から取り出した後、空気はサンプルが戻ってその前の寸法を縮小するように、48時間暗条件下で膨潤したポリマー試料を乾燥させます。 </li><li>任意の表面に結合した物質や残留溶媒を除去するために、脱イオン水で十分にQD-組み込まれたサンプルを洗ってください。 </li></ol></li></ol>ナノ粒子の3可視化は、PDMSにカプセル化スウェル<ol><li> シリコーンサンプル調製： <ol><li>新鮮なメスの刃（5.5ミリメートル×11 mm）の2つのシリコーンの正方形を切り取ります。これはシリコーンサンプルの内部表面を露出させていることを確認してください。 </li></ol></li><li> 蛍光イメージング： <ol><li> microscoにシリコーンサンプルを置きますポリマーの新たにカット側はスライドガラスに完全に密着していることを保証するイメージングのためのPEのスライド、。顕微鏡スライドとの円滑な接触を確保するために軽くシリコーン部分を押し下げます。顕微鏡のステージ上にサンプルを置きます。 </li><li> 20 MHzまたは類似の速度で5ピコ秒のパルスからなる488nmの分光濾過レーザー線を使用して完全な寿命の蛍光測定値23 488 ​​を生成するために、レーザ出力の直接結合、音響光学可変フィルタシステムを使用してnmレーザーライン。 10X対物レンズの背面開口部にダイクロイックミラー（488 nm）ので反射してサンプルの上にされ、特注のレーザースキャニングユニットを（補足情報を参照してください）​​、使用したレーザー光の焦点を合わせます。 <ol><li>その後、同じダイクロイックミラーを透過していることを、同じ目的を持つ蛍光発光を収集します。単一光子計数モードで動作するアバランシェフォトダイオードに向けて、この光を向けます。プロセスlifeti<html>時間相関単一光子計数（TCSPC）ボードを使用して私の測定。23 </li></ol></li><li>開始時の機器の応答関数（IRF）と各実験セッションの終了を記録します。23 注：TCSPC実験で記録された信号は、検出器における光子の到着とその後のレーザーパルスの生成との間の時間遅延を示すべきです。この時間遅延は、しかしながら、測定装置のIRFと畳み込みされるべきです。したがって、IRFは、楽器の応答と比較して有意に短い寿命（〜100ピコ秒）を持つ100 nMのオーラミンO、に機器の応答として測定されます。 </li><li>蛍光強度の指数関数的減衰の非線形フィッティングにより寿命を抽出し、最尤推定量（MLE）アルゴリズム25-27を使用して、IRFからこれをデコンボリューション。 MLE（γjは ）のように計算されます eq1.jpg &quot;/&gt; N iは 、チャネルIでの光子カウント数であり、kは各蛍光減衰のためのチャネル（またはビン）の数であり、pはI（j）は 、光子のグループは、粒子が持っている私場合、チャネルに分類する確率であります生涯jは 、Nは与えられた減衰のためのカウントの総数です。 </li><li>サイズ512×512ピクセルと264の画像からなる連続スキャンを作成し、5分間、各サンプルの蛍光強度と寿命のデータを記録します。 150光子とプロセスのしきい​​値と計算されたMLEこの使用して、MATLABで、2次元の蛍光強度、寿命や強度加重寿命マップを提供するために、これらを組み合わせます。 </li></ol></li></ol>

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C., 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=Fluorescence+detection+methods+for+microfluidic+droplet+platforms.">Fluorescence detection methods for microfluidic droplet platforms.</a> J. Vis. Exp. (58), e3437 (2011).</li><li>K&#246;llner, 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=Fluorescence+pattern+recognition+for+ultrasensitive+molecule+identification:+comparison+of+experimental+data+and+theoretical+approximations.">Fluorescence pattern recognition for ultrasensitive molecule identification: comparison of experimental data and theoretical approximations.</a> Chem. Phys. Lett. 250 (3-4), 355-360 (1996).</li><li>K&#246;llner, M., Wolfrum, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+photons+are+necessary+for+fluorescence-lifetime+measurements.">How many photons are necessary for fluorescence-lifetime measurements.</a> Chem. Phys. Lett. 200 (1-2), 199-204 (1992).</li><li>Edel, J. B., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Single+Molecule+FRET+Efficiency+Determination+for+Surface+Immobilized+DNA+Using+Maximum+Likelihood+Calculated+Lifetimes.">Accurate Single Molecule FRET Efficiency Determination for Surface Immobilized DNA Using Maximum Likelihood Calculated Lifetimes.</a> J. Phys. Chem., B. 111 (11), 2986-2990 (2007).</li><li>Bear, J. C., 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=Doping+Group+IIB+Metal+Ions+into+Quantum+Dot+Shells+via+the+One-Pot+Decomposition+of+Metal-Dithiocarbamates.">Doping Group IIB Metal Ions into Quantum Dot Shells via the One-Pot Decomposition of Metal-Dithiocarbamates.</a> Adv. Opt. Mater. 3 (5), 704-712 (2015).</li><li>Li, L., Hu, J., Yang, W., Alivisatos, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Band+Gap+Variation+of+Size-+and+Shape-Controlled+Colloidal+CdSe+Quantum+Rods.">Band Gap Variation of Size- and Shape-Controlled Colloidal CdSe Quantum Rods.</a> Nano Letters. 1 (7), 349-351 (2001).</li></ol>

The authors have nothing to disclose.

Cross-sectional fluorescence imaging allows for direct visualization of nanoparticles during swell encapsulation. The kinetics of encapsulation has been shown, with the drive toward a high nanoparticle surface concentration demonstrated. The extent of nanoparticle incorporation is shown to vary with swell encapsulation time (described in section 2.3), with the total amount of incorporated nanoparticles increasing as this time is extended, with the particle concentration localized at the surface if the polymer samples are...

ナノ材料の適用は、長い間、新規技術への関心の高まりの面積を務めています。1-3。これは、化粧品、衣類、包装、エレクトロニクスなどの日用品、中のナノ粒子の成長の使用を含んでいた。4-6ナノ粒子を用い向けて主要なドライブ機能材料に、7つ別の利点は、ホストマトリックスに重要な特性を導入する、容易に複合材料を形成する能力である。粒径の変動によって調整プロパティする能力に加えて、材料への高い反応性の相対由来など触媒機能、材料の強化および電気的特性のチューニング。8-12 ナノ粒子-高分子複合材料は、ホストマトリックスの製造中に所望のナノ粒子の直接積分である最も単純なそれらの技術の範囲を介して達成することができる。13,14これR全体にナノ粒子材料の均等間隔で均質な材料でesults。しかし、多くのアプリケーションでは、ナノ複合材料の外部インタフェースに存在する活性物質を必要とします。材料のバルクを通じて多くのナノ粒子の廃棄物が存在する結果として、直接の取り込みは、時には高価なナノ粒子材料の効率的な使用にはなりません。15,16を直接取り込みを達成するために、ナノ粒子はまた、ホストマトリックスの形成と互換性がなければなりません。これは、特に、一般的に高活性なナノ粒子によって影響を受ける可能性がある金属錯体触媒機構によって促進される熱硬化性ポリマーの場合のように多面的な反応を必要とする合成に、挑戦することができる。14 ポリマー合成中に直接ナノ粒子の取り込みに関連したかなりの欠点は、ナノ粒子incorporatiを制限することを目的とした技術の開発につながっています表面層に上。17-21スウェルカプセル化は、ポリマーのバルク中の限られた無駄で、高い表面のナノ粒子の濃度を達成するために、文献で ​​報告された最も成功した戦略の一つである。17-19技術は、ポリマーの溶剤駆動膨潤を利用します行列は、分子種とナノ粒子の侵入を可能にします。膨潤溶媒を除去すると、マトリックス内の種は表面に局在する種の最高濃度で、所定の位置に固定となります。現在までに、うねりカプセル化の報告用途のほとんどは、活性剤は、材料表面であることが鍵である抗菌ポリマーの製造、に向けられています。これらのレポートの多くが強化された抗菌活性を示しているが、正確な表面ナノ粒子組成物はほとんど詳細に精査されていません。クリックらは最近、重要なINSIを提供し、ナノ粒子の侵入の直接可視化するための方法を実証しましたうねりのカプセル化によって達成動態および表面のナノ粒子の濃度にGHT。22 この作品は、セレン化カドミウム量子ドット（QD）、ポリジメチルシロキサン（PDMS）と蛍光イメージングを使用して、それらの組み込みの直接可視化へのそれらのうねりのカプセル化の合成を詳述します。膨潤溶液中で膨潤カプセル化時間及びナノ粒子の濃度を変化させることの効果が検討されています。蛍光可視化技術は、PDMS中にナノ粒子侵入の直接的なイメージングを可能にし、QDの最高濃度は、材料の表面であることを示しています。

<table><tbody><tr><td>Polydimethylsiloxane sheets</td><td>NuSil</td><td>-</td><td>Medical Grade</td></tr><tr><td>Oleylamine</td><td>Sigma Aldrich</td><td>O7805</td><td>Technical Grade</td></tr><tr><td>Trioctylphosphine</td><td>Sigma Aldrich</td><td>117854</td><td>Technical Grade</td></tr><tr><td>Trioctylphosphine oxide</td><td>Sigma Aldrich</td><td>346187</td><td>Technical Grade</td></tr><tr><td>1-Octadecene</td><td>Sigma Aldrich</td><td>O806</td><td>Technical Grade</td></tr><tr><td>Zinc diethyldithiocarbamate</td><td>Sigma Aldrich</td><td>329703</td><td>-</td></tr><tr><td>Oleic acid</td><td>Sigma Aldrich</td><td>364525</td><td>Technical Grade</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>471283</td><td>-</td></tr><tr><td>Cadmium oxide</td><td>Alfa Aesar</td><td>33235</td><td>-</td></tr><tr><td>Hexadecylamine</td><td>Alfa Aesar</td><td>B22459</td><td>Technical Grade</td></tr><tr><td>1-Dodecylphosphonic acid</td><td>Alfa Aesar</td><td>H26259</td><td>-</td></tr><tr><td>Selenium powder</td><td>Acros</td><td>19807</td><td>-</td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>366919</td><td>-</td></tr><tr><td>n-Hexane</td><td>Sigma Aldrich</td><td>208752</td><td>-</td></tr><tr><td>Microscope slides</td><td>VWR</td><td>631-0137</td><td>Thickness No. 1</td></tr></tbody></table>

advanced compositional analysis of nanoparticle-polymer composites using direct fluorescence imaging

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials.

600nm程度のラムダ最大で、赤色蛍光を示した。22,28赤色発光量子ドットはサイズ寸法強い閉じ込め政権内にある量子ロッドによって励起子の閉じ込めによるものでした。 Li らは、量子ロッドのため、発光シフト幅またはロッドの長さのいずれかの増加に伴ってエネルギーを低下させることを示しました。彼らはさらに、放出は、主ロッドの幅は、それ?...

Watch this Scientific Journal Video about Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging at JoVE.com

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

直接蛍光イメージングを用いたナノ粒子 - ポリマー複合材料の高度な組成分析

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise ...

advanced-compositional-analysis-nanoparticle-polymer-composites-using

Research

JoVE Journal

Engineering

7.7K ビュー.  Imperial College London. Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

ビデオ: Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius&#160;and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9.&#160;Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37.&#160;In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.

Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.

監禁静止と流れるコロイド·ポリマー混合物の共焦点イメージング

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed ...

化学

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

組合せの合成と高分子膜とナノ粒子のライブラリからのハイスループットタンパクリリース

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

生物工学

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step process that finishes with Negishi coupling. Negishi coupling is a cross-coupling process in which a chemical precursor is first lithiated, followed by transmetallation with ZnCl2. The resultant organozinc compound can be coupled to a dibrominated aromatic precursor to give the conjugated monomer. Polymer films can be prepared via electropolymerization of the monomer and characterized using cyclic voltammetry and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. Nanoparticles (NPs) are prepared via emulsion polymerization of the monomer using a two-surfactant system to yield an aqueous dispersion of the polymer NPs. The NPs are characterized using dynamic light scattering, electron microscopy, and UV-Vis-NIR-spectroscopy. Cytocompatibility of NPs is investigated using the cell viability assay. Finally, the NP suspensions are irradiated with a NIR laser to determine their effectiveness as potential materials for photothermal therapy (PTT).

A protocol is presented for the synthesis and preparation of nanoparticles consisting of electroactive polymers.

電気活性ポリマーナノ粒子出展光熱プロパティ

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step ...

工学

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

制御された合成および均一性の高いポリの蛍光トラッキング（<em&gt; N</em&gt; -isopropylacrylamide）ミクロゲル

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

単一分子レベルでの環状ポリマーの合成と溶融状態におけるそれらの拡散運動のキャラクタリゼーション

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

トランスミッションベースのノマルスキー型差動干渉造影顕微鏡を用いたプラズモニックナノ粒子の分光法の実行

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

ナノスケール赤外分光法による多相高分子システムの探索

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition is commonly assessed using infrared spectroscopy, which provides an average fingerprint of the various materials contained in the volume probed. However, this approach does not offer any details on the arrangement of the phases in the material. Interfacial regions between two polymeric phases, often in the nanoscale range, are also challenging to access. Photothermal nanoscale infrared spectroscopy monitors the local response of materials excited by infrared light with the sensitive probe of an atomic force microscope (AFM). While the technique is suitable for interrogating small features, such as individual proteins on pristine gold surfaces, the characterization of three-dimensional multicomponent materials is more elusive. This is due to a relatively large volume of material undergoing photothermal expansion, defined by the laser focalization onto the sample and by the thermal properties of the polymeric constituents, compared to the nanoscale region probed by the AFM tip. Using a polystyrene (PS) bead and a polyvinyl alcohol (PVA) film, we evaluate the spatial footprint of photothermal nanoscale infrared spectroscopy for surface analysis as a function of the position of PS in the PVA film. The effect of the feature position on the nanoscale infrared images is investigated, and spectra are acquired. Some perspectives on the future advances in the field of photothermal nanoscale infrared spectroscopy are provided, considering the characterization of complex systems with embedded polymeric structures.

著者スポットライト:Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples.

多相高分子系を探索するためのナノスケール赤外分光法の進歩

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

個々のリポソーム間の組成不均一性を検出するための定量的蛍光顕微鏡ベースの単一リポソームアッセイ

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

直接蛍光イメージングを用いたナノ粒子 - ポリマー複合材料の高度な組成分析

要約

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監禁静止と流れるコロイド·ポリマー混合物の共焦点イメージング

組合せの合成と高分子膜とナノ粒子のライブラリからのハイスループットタンパクリリース

電気活性ポリマーナノ粒子出展光熱プロパティ

制御された合成および均一性の高いポリの蛍光トラッキング（

単一分子レベルでの環状ポリマーの合成と溶融状態におけるそれらの拡散運動のキャラクタリゼーション

トランスミッションベースのノマルスキー型差動干渉造影顕微鏡を用いたプラズモニックナノ粒子の分光法の実行

多相高分子系を探索するためのナノスケール赤外分光法の進歩

多相高分子系を探索するためのナノスケール赤外分光法の進歩

多相高分子系を探索するためのナノスケール赤外分光法の進歩

個々のリポソーム間の組成不均一性を検出するための定量的蛍光顕微鏡ベースの単一リポソームアッセイ