著者らは、Scott HaardとAbigail Marinackがこの作業の全般的なサポートと調整について認めてくれるだろう。

ヒト尿サンプルを含むすべての研究は、Pharmasan / Neuroscience Institutional Review Board（IRB）によって承認された手順に従って実施された。 1.尿試料採取およびクレアチニン（Cr）測定<ol><li>次の朝の尿サンプル5mLをca.保存まで3N HCl250μLを含む5mL輸送管Aで一晩10時間絶食させ、使用するまで-20℃で保存する。 </li><li>解凍しボルテックスする5mLの輸送尿チューブと遠心分離機（ 例えば Sorvall）（2297xg、10分）中で遠心分離する。 </li><li> 5mLの輸送管Aから2回、1mLの尿を分注する。 </li><li>尿クレアチニン法によるCrの測定26 。 </li></ol>標準、ISおよび品質管理（QC）サンプルの調製<ol><li> 0.01％HCOOH移動相A（MA）を含む水中で100μg/ mLの3-NTのストック溶液を調製し、-20℃で保存する。 </li><li>メイク100μg/ mLの3-NTストック溶液をMAで希釈することにより、100ng / mLの濃度の3-NT作動標準溶液。 </li><li>ブランクおよび二重ブランク試料とともに、3-NTを含まない0.15M HCl酸性ブランク尿で100ng / mLの作業標準液を希釈することにより、5〜2500pg / mLの標準物質を生成する。 </li><li> -20℃でMAとストアと水で100μg/ mLのにある13 C 9 -3-NTのストック溶液を調製します。 </li><li> 100μg/ mLの13 C 9 -3-NTの希釈により500 pg / mlでで動作している溶液を作るには、MAとのストック溶液です。 </li><li>酸性化されたブランク尿中の3-NT作業標準の希釈によって、LLOQ、低、中および高レベル（ すなわち 、10,25,100,500および1,250 pg / mL）をカバーする5レベルのQCサンプルを確立する。 </li></ol>固相抽出手順<ol><li>解凍し、尿サンプル、標準品およびQCサンプルをボルテックスする。 </li><li>尿サンプル、標準およびQCサンプルを追加les（各250μL）を32ウェルの清潔な2mL 96ウェル収集プレートに移した。 </li><li>二重ブランク試料ウェルを除く各ウェルに500 pg / mL IS作動液（50μL）を導入する。二重ブランク試料ウェルに0.01％HCOOH（50μL）を添加する。 </li><li> 0.1％HCOOH（250μL）を含むLC-MS / MS水を加える。 </li><li>上記の混合物を8チャンネルピペットで3回混合する。 </li><li> SPE装填までプレートを覆う。 </li><li> MCX 96ウェル抽出プレートとコレクションリザーバーを陽圧プロセッサーに置きます。 </li><li>抽出プレートを、流出するMeOH（200μL）で吸着剤に通す。 </li><li>吸着剤に2％HCOOH（200μL）を流して平衡させる。 </li><li>予め混合した各サンプルの全容積を、8チャンネルのピペットで慎重に予備条件抽出プレートにロードする。 </li><li>混合物を流れさせるために陽圧プロセッサーに低い正の圧力（ 例えば 3psi）を設定する吸収剤をゆっくりと摂取し、必要に応じて圧力を調整します。 </li><li> 2％HCOOH（200μL）を含む水を吸着剤に流してウェルを洗浄する。 </li><li>吸着剤にメタノール（200μL）を流してウェルを洗浄する。 </li><li>吸着剤に水（200μL）を流してウェルを洗浄する。 </li><li>ポジティブプレッシャープロセッサーで、正の高圧力設定（ 例えば 、40 psi）でウェルを完全に乾燥させます。 </li><li>リザーバーを清潔な2 mL 96ウェルコレクションプレートに交換します。 </li><li>抽出プレートから保持された検体およびISを溶出するために、pH 9（50μL）で25mM NH 4 OAcを適用する。 </li><li>溶出液を中和するために、5％HCOOH（50μL）を含むLC-MSピペット水。 </li><li> 8チャンネルピペットで3回ミックスし、分析のためにLC-MS / MSステーションに提出する。 </li></ol> LC-MS / MS分析<ol><li>メスシリンダーで2000mLのLC-MS水を正確に測定し、2Lボトルに移します。 </li><li> P200μLの純粋なHCOOHを上記のLC-MS水を含むボトルに入れる。 </li><li>完全に混合し、移動相A（MA）とラベルする。イニシャル、準備日、有効期限を記入してください。 </li><li> 2リットルのLC-MSメタノールボトルに入れ、開始日と有効期限のある移動相B（MB）とラベルを付けます。 </li><li>オートサンプラーの温度を4℃に設定します。 </li><li>調製したサンプルを含むコレクションプレートをオートサンプラにセットします。 </li><li>オーブンにPFPカラム（150 mm x 2.1 mm id、3μm）とガードカラムを置きます。 </li><li>オーブンの温度を30℃に設定します。 </li><li> 表1に示すLCグラジエント溶出を用いた取得法を用いて10分間平衡化する。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 時間（分） </td><td> モジュール </td><td> イベント </td><td> パラメータ </td></tr><tr><td> 0 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 5 </td></tr><tr><td> 0.5 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 20 </td></tr><tr><td> 1 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 50 </td></tr><tr><td> 3 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 80 </td></tr><tr><td> 4 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 90 </td></tr><tr><td> 4.01 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 95 </td></tr><tr><td> 5.5 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 95 </td></tr><tr><td> 5.6 </td><td>パンプス</td><td>ポンプBコンク。 </td><td> 5 </td></tr><tr><td> 7 </td><td>コントローラ</td><td>やめる</td><td></td></tr></tbody></table> 表1： 液体クロマトグラフィーグラジエント溶出条件 <ol start="10"><li>以下を含むバッチリストを作成するスタンダード、QCおよび尿サンプル。 </li><li>調製したサンプル（12μL）の注入によりバッチを開始する。 </li></ol> 5.ピーク識別、統合、データ処理<ol><li>ソフトウェアを使用したデータの取得と処理を制御します。 </li><li>すべてのサンプルについて3-NTピークとISピークを特定し、統合します。 </li><li> 3-NTおよびISのピーク面積比対公称3-NT濃度の1 / xの重み付け係数を線形回帰することにより、3-NT定量のために10-2,500 pg / mLの範囲の標準曲線を確立する。 </li><li>標準曲線を使用してすべてのサンプルを定量する。 </li><li> QCサンプルが確立された範囲内にあるかどうかを決定する。 </li><li>検出された尿サンプルの濃度をnMまたはnmol / mmol Crの単位で最終結果に変換します。 </li></ol>

<ol>
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著者らは、彼らには利益相反がないと宣言している。

以前にヒトの尿サンプル中の内因性遊離3-NTについての文献に報告された濃度の実質的なバリエーションが利用可能なアッセイ8、9、10、11に関連した方法論の問題点を明らかにする。ヒト尿中の3-NTの低い基底レベルの正確な決定は、試料調製およびLC-MS / MS分析のための特別な予防措置を必要とす?...

酸化ストレスが臨床上のプレゼンテーションに及ぼす影響は、近年ますます重要視されています1 。探索されているバイオマーカーの1つは、3-ニトロチロシン（3-NT）であり、反応性窒素種（RNS）がチロシン、カテコールアミン神経伝達物質前駆体と相互作用するときに生成する最終安定生成物である。 3-NTは、in vivoでの RNSのためのバイオマーカーとして臨床的価値を有していてもよいが、チロシンの性質及び機能の実質的な変化が悪影響を対応するタンパク質及び細胞機能1,2に影響を及ぼし得ます。新興の研究では、3-NTは、炎症状態で3、神経変性疾患の重要な役割を果たしていることを示唆している4、5、6心血管疾患や糖尿病7と同様に酸化ストレスに関連する状態。しかし、これらのobservations、感度及び/又は選択8、9、10、11に欠ける方法からの結果に基づいています。以前に文献に報告された生​​物学的サンプルの3-NTの濃度範囲の大部分は、深刻な分析上の問題がこれらのアッセイに関連していることを示しており、3-NTレベルを正確に定量化し、 。 生物学的マトリックス中の自由3-NTの定量化は、人間と機器8、9、10、11に特別な挑戦を提示しています。第1に、内因性3-NTのトレースレベルは、超高感度検出を必要とする。第二に、多数の構造的に類似した類似体、特にチロシンが存在し、これは非常に過剰であり、高度の選択性を必要とする。第3に、遍在する硝酸塩および亜硝酸塩によるチロシンニトロ化による3-NTの人工的形成は、3-NTの誤った過大評価を避けるために試料調製中に特別な配慮を必要とする。 3-NTを測定するために用いられる方法の多種多様な中でも、MS / MSは、その優れた感度と選択性11、12、13、14に金標準的な方法を考えてきました。ガスクロマトグラフィー（GC）は、MS / MSは、最良の感度を提供結合、しかし、必須サンプル誘導体化ステップは、臨床的有用性15、16のために効率的であることがあまりにも面倒で時間がかかります。 LC-MS / MSは複雑なサンプルの誘導体化を必要とせず、より有望な選択肢になります。それにもかかわらず、克服すべきいくつかの障害があります文献に報告されたLC-MS / MS法のnsitivity低豊富3-NT 7、17、18の測定のために改善する必要があり、比較的長いターンアラウンドタイムは、ハイスループットアプリケーション12のために短くする必要があり、13、17、19 。 さらに、臨床応用を考慮するとき、使用される生物学的マトリックスは重要な役割を果たす。それは簡単で得ることが安価で可能な20、21、22であれば、非侵襲性であるべきです。文献で伝統的に使用されている血漿である血漿は、臨床的に望ましいマトリックスではないので、非侵襲的かつ費用対効果の高い尿を利用する方法論が求められていた。 relを開発するためのいくつかの試みiableと特定LC-MS / MS方法論は、尿9、10、11を用いて行われてきました。しかし、それらはすべて、臨床的使用のために十分に選択的、信頼できる、または効率的であるのに足りない。 3-NTの分析のためのサンプルクリーンアップが疑問視されていると強カチオン交換（SCX）と逆相C18-OHの順次SPE 6提案されているような従来の逆相カートリッジ（C18型）を用いて、優勢なSPEの有効性、 7、19。最近開発されたLC-MS / MS法は、手動C18 SPE、分取高圧液体クロマトグラフィー（HPLC）、および3-NT 23の分析のためのオンラインSPEの多段階精製プロセスを利用した。この方法は、0.041nMのLLOQを有する臨床目的のために十分に敏感であったが、クリーンアッププロセスは集中的で面倒であり、赤色の3 mLの尿。高スループットの実現可能性が制限されています。クリーンアッププロセス14の効率を改善するために、分子的にインプリントされたポリマーをSPE吸着剤として使用したが、得られたLLOQ（0.7μg/ mL）は臨床検体にとって十分に低いものではなかった。別の方法は、0.022 nMで24の検出限界（LOD）を達成するために、2次元（2D）LC-MS / MSおよびサンプルクリーンアップのためのイムノアフィニティークロマトグラフィーを必要としました。これらの方法はすべて3-NTの評価において進歩を遂げてきましたが、臨床応用に必要な感度、信頼性、効率を達成したものはありませんでした。 3-NTの病態とその臨床的環境における酸化ストレスのバイオマーカーとしての役割を調べるために、我々はハイスループットの臨床応用が可能なシンプルで効率的で正確で正確な方法論を開発した25 。小型化された混合モードカチオンexc誘導体化、蒸発および2D-LCを必要とする既存の方法に見られる欠点を迂回して、単一の抽出で3-NTの簡便で効果的なサンプルクリーンアップおよび濃縮を達成するためにハンゲイ（MCX）96ウェル抽出マイクロプレートを実施した。移動相における添加剤として0.01％HCOOHを用いた液体クロマトグラフィーは、迅速なサイクル時間で増強されたシグナル応答を提供した。選択性は、3-NTの選択的溶出のための軽度のNH 4 OAc溶出溶液の適用、および3-NTおよび内部標準（IS）の両方のMRM転移の使用によってさらに改善された。マトリックス効果は、定量化のために好ましい量の13 C標識同位体ISを使用することによって補償された。この方法論の出現により、研究者および臨床医は、臨床状態における3-NTの役割を検証し、さらに酸化ストレスの影響を探ることができるようになる。

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integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

遊離3-ニトロチロシン（3-NT）は、酸化ストレスのための可能なバイオマーカーとして広範に使用されている。広範な病理学的状態において、3-NTのレベルの上昇が報告されている。しかし、既存の方法は、3-NTの低内在レベルを確実に測定するのに必要な十分な感度および/または特異性を欠いており、臨床応用には扱いにくい。したがって、3-NTのレベルを正確に定量化し、病理学的状態における3-NTの役割を確認するためには、分析的改善が急務である。このプロトコルは、非侵襲性バイオマーカーとしてのヒト尿中の3-NTの迅速かつ正確な測定のための小型液体クロマトグラフィータンデム質量分析（LC-MS / MS）検出と小型化固相抽出（SPE）酸化ストレス96ウェルプレートを使用するSPEは、退屈な誘導体化および蒸発工程なしでサンプルのクリーンアップおよび分析物の濃縮を組み合わせることによりプロセスを顕著に単純化した廃棄物処分、汚染の危険性および全体的な処理時間を削減する。 SPE溶出溶液としてのpH9での25mM酢酸アンモニウム（NH 4 OAc）の使用は、選択性を実質的に向上させた。質量分析法のシグナル応答は、多重反応モニタリング（MRM）移行の調整によって改善されました。ペンタフルオロフェニル（PFP）カラム（150mm×2.1mm、3μm）上の添加剤として0.01％HCOOHを使用すると、シグナル応答がさらに2.5倍改善され、全実行時間が7分に短縮されました。 10pg / mL（0.044nM）の定量下限（LLOQ）が達成され、報告されたアッセイより有意な感度の改善を示した。この単純化された迅速で選択的で敏感な方法により、尿試料の2つのプレート（n = 192）を24時間の時間内で処理することが可能になる。顕著に改善された分析性能、および非侵襲的かつ安価な尿サンプリングを考慮すると、提案されたアッセイは、前臨床および臨床研究。

図1は、3-NTが、これらの非常に過剰な化合物に起因する共溶出妨害を排除し、結果としてアッセイ選択性の程度を高める、最適化されたLC条件下で他の構造的に類似のチロシン類似体から完全にクロマトグラフィーで分離されることを示す。さらに、0.45mL /分の流速でのMAおよびメタノール中の添加剤としての0.01％HCOOHによる勾配溶出により、3-NT...

Watch this Scientific Journal Video about Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications at JoVE.com

臨床応用のためのヒト尿中の 3-ニトロチロシンの小型固相抽出と LC-MS/MS 検出の統合 |化学 |ビデオ

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

混合モードカチオン交換（MCX）96ウェルマイクロプレート上での効率的な固相抽出と組み合わせた選択的かつ高感度の液体クロマトグラフィータンデム質量分析（LC-MS / MS）法を、遊離3-ニトロチロシンの測定のために開発した3-NT）をヒト尿中に高スループットで導入することができ、これは臨床用途に適している。

臨床応用のためのヒト尿中の3-ニトロチロシンの小型化固相抽出およびLC-MS / MS検出の統合

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

integration-miniaturized-solid-phase-extraction-lc-msms-detection-3

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9.2K ビュー.  Pharmasan Labs, Inc.. 混合モードカチオン交換（MCX）96ウェルマイクロプレート上での効率的な固相抽出と組み合わせた選択的かつ高感度の液体クロマトグラフィータンデム質量分析（LC-MS / MS）法を、遊離3-ニトロチロシンの測定のために開発した3-NT）をヒト尿中に高スループットで導入することができ、これは臨床用途に適している。 

ビデオ: Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

プラズマを介した疎水性有機金属フレームワークの製造は、アンモニアの除去のためにペルフルオロアルカンの化学的気相成長法を強化

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

化学

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

分子、イオン、および音響キャビテーションと固体粒子を活性化

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

固相樹脂系の方法論を経由して、非対称フェロセン派生バイオコンジュゲートシステムのための合成方法論

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

保存科学における粉末X線回折：遺産アートオブジェクトに対する腐食製品のルーチンの結晶構造の決意に向けて

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

動的光散乱顕微鏡による濁りソリューションにおける粒度分布の測定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

2 &#39;末端を含むRNAのオリゴマーの固相合成のためのプロトコール<em&gt; O</em&gt;チオフェンメチル修飾と環状二色性によるキャラクタリゼーション

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

抽出と神経伝達物質カテコールアミン及びその代謝物の高速液体クロマトグラフィーによる分析のための便利な方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Surface Properties of Synthesized Nanoporous Carbon and Silica Matrices

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) with a 4.6 nm pore size, and ordered silica porous matrix, SBA-15, with a 5.3 nm pore size. This work describes the surface properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the differently ordered porous materials with similar pore sizes. For this purpose, OMC and SBA-15 with highly ordered nanoporous structures are synthesized via impregnation of the silica matrix by applying a carbon precursor and by the sol-gel method, respectively. The porous structure of investigated systems is characterized by an N2 adsorption-desorption analysis at 77 K. To determine the electrochemical character of the surface of synthesized materials, potentiometric titration measurements are conducted; the obtained results for OMC shows a significant pHpzc shift toward the higher values of pH, relative to SBA-15. This suggests that investigated OMC has surface properties related to oxygen-based functional groups. To describe the surface properties of the materials, the contact angles of liquids penetrating the studied porous beds are also determined. The capillary rise method has confirmed the increased wettability of the silica walls relative to the carbon walls and an influence of the pore roughness on the fluid/wall interactions, which is much more pronounced for silica than for carbon mesopores. We have also studied the melting behavior of D2O confined in OMC and SBA-15 by applying the dielectric method. The results show that the depression of the melting temperature of D2O in the pores of OMC is about 15 K higher relative to the depression of the melting temperature in SBA-15 pores with a comparable 5 nm size. This is caused by the influence of adsorbate/adsorbent interactions of the studied matrices.

Here we report the synthesis and characterization of ordered nanoporous carbon (with a 4.6 nm pore size) and SBA-15 (with a 5.3 nm pore size). The work describes the surface and textural properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the materials.

合成多孔性カーボンとシリカ マトリックスの表面特性

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

[4.4] スピロ エナンチオマーの固相合成

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

LC-MS/MS 診断フラグメンテーションによる自然な製品の発見: ミクロシスチン分析のためのアプリケーション

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...