我々は感謝してDARPA主催のマイクロ/ナノ流体の基礎フォーカス（MF3）契約番号N66001-10-1-4003、下のセンターからDARPAの助成金N660001-09-C-2082からの資金調達を認める。

<ol>
	<li>Jung, B., Bharadwaj, R., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+millionfold+sample+stacking+using+transient+isotachophoresis.">On-chip millionfold sample stacking using transient isotachophoresis.</a> Anal. Chem. 78 (7), 2319-2319 (2006).</li><li>Jung, B., Zhu, Y., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+100+am+fluorophores+using+a+high-sensitivity+on-chip+CE+system+and+transient+isotachophoresis.">Detection of 100 am fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis.</a> Anal. Chem. 79 (1), 345-345 (2007).</li><li>Everaerts, F. M., Beckers, J. L., Verheggen, T. P. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotachophoresis:+Theory,+instrumentation,+and+applications.">Isotachophoresis: Theory, instrumentation, and applications.</a> Elsevier Science &#38; Technology. , (1976).</li><li>Khurana, T. K., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconcentration,+separation,+and+indirect+detection+of+nonfluorescent+analytes+using+fluorescent+mobility+markers.">Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers.</a> Anal. Chem. 80 (1), 279-279 (2008).</li><li>Bercovici, M., Kaigala, G. V., Backhouse, C. J., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carrier+ampholytes+assay+for+portable,+label-free+detection+of+chemical+toxins+in+tap+water.">Fluorescent carrier ampholytes assay for portable, label-free detection of chemical toxins in tap water.</a> Anal. Chem. 82 (5), 1858-1858 (2010).</li><li>Bercovici, M., Kaigala, G. V., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+analyte+identification+using+isotachophoresis+and+a+fluorescent+carrier+ampholyte+assay.">Method for analyte identification using isotachophoresis and a fluorescent carrier ampholyte assay.</a> Anal. Chem. 82 (5), 2134-2134 (2010).</li><li>Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+system+for+isotachophoresis+assays.">Miniaturized system for isotachophoresis assays.</a> Lab Chip. 10 (17), 2242-2242 (2010).</li><li>Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantification+of+isotachophoresis+zones+using+nonfocusing+fluorescent+tracers.">Imaging and quantification of isotachophoresis zones using nonfocusing fluorescent tracers.</a> Anal. Chem. 81 (8), 3022-3022 (2009).</li><li>Schoch, R. B., Ronaghi, M., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+selective+extraction,+isolation,+preconcentration,+and+quantitation+of+small+RNAs+from+cell+lysate+using+on-chip+isotachophoresis.">Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.</a> Lab Chip. 9 (15), 2145-2145 (2009).</li><li>Persat, A., Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+from+whole+blood+using+isotachophoresis.">Purification of nucleic acids from whole blood using isotachophoresis.</a> Anal. Chem. 81 (22), 9507-9507 (2009).</li><li>Bercovici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+urinary+tract+infections+using+isotachophoresis+and+molecular+beacons.">Rapid detection of urinary tract infections using isotachophoresis and molecular beacons.</a> Anal. Chem. 83 (11), 4110-4110 (2011).</li><li>Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+malaria-infected+erythrocytes+using+isotachophoresis.">Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.</a> Anal. Chem. 83 (24), 9715-9715 (2011).</li><li>Persat, A., Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+electrolyte+chemistry+for+microfluidic+electrokinetics.+Part+I:+Acid-base+equilibria+and+pH+buffers.">Basic principles of electrolyte chemistry for microfluidic electrokinetics. Part I: Acid-base equilibria and pH buffers.</a> Lab Chip. 9 (17), 2437-2437 (2009).</li><li>Garcia-Schwarz, G., Bercovici, M., Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+dispersion+in+isotachophoresis.">Sample dispersion in isotachophoresis.</a> J. Fluid Mech. 1 (1), 1-1 (2011).</li><li>Kohlrausch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+concentrations-verschiebungen+durch+electrolyse+im+inneren+von+l&#214;sungen+und+l&#214;sungsgemischen.">&#220;ber concentrations-verschiebungen durch electrolyse im inneren von l&#214;sungen und l&#214;sungsgemischen.</a> Ann. Phys. 298 (10), 209-209 (1897).</li><li>Alberty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+boundary+systems+formed+by+weak+electrolytes.+Theory+of+simple+systems+formed+by+weak+acids+and+bases.">Moving boundary systems formed by weak electrolytes. Theory of simple systems formed by weak acids and bases.</a> J. Am. Chem. Soc. 72 (6), 2361-2361 (1950).</li><li>Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+zone+electrophoresis.+I.+Steady-state+moving-boundary+systems+formed+by+different+electrolyte+combinations.">Multiphasic zone electrophoresis. I. Steady-state moving-boundary systems formed by different electrolyte combinations.</a> Biochem. 12 (5), 871-871 (1973).</li><li>Persat, A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling+by+simultaneous+selective+isotachophoresis+and+hybridization+with+molecular+beacons.">MicroRNA profiling by simultaneous selective isotachophoresis and hybridization with molecular beacons.</a> Anal. Chem. , (2011).</li></ol>

我々は、開示することは何もありません。

ITP法は、迅速に敏感な、堅牢な検出およびイオン分子の処理を有効にするここで提示した。 ITPを使用する際の主な課題は、LEおよびTEバッファの適切な選択です。それは非常に高い絶対的な移動度を持つ強さの酸であるため、非常に予測可能な性質を持っているのでアニオンITPで、我々は一般的に主要な陰イオンとして塩化物を選択します。従って、アニオン性ITPのバッファの選択は、通常、適切なTEと対を選択することに低減されます。選択的な焦点の実験では、TEの選択は、汚染イオンの排除に重要です。汚染物質が存在しないアプリケーションでは、低TE実効移動度は、焦点の速い速度につながります。 MES、MOPS、HEPES、トリシン：我々は相対的にアニオン性のTEイオンを行儀には、次の手順を使用することをお勧めします。対イオンの選択は、システムのpHとTE効果的なモビリティの両方に影響を与えます。一般的な対イオンは、トリス（pKaは8.1）、ビス - トリス（pKaは6.4）です。低い、または高いpHを必要とするアッセイのために、我々はピリジン（pKaは5.25）をお勧めしそれぞれエタノールアミン（pKaは9.5）。 表1と表2に、アニオン性およびカチオン性ITPに対して、それぞれ、我々はいくつかの有用な緩衝液の例をまとめたものです。我々は、カチオン性LEイオン等のアニオン性LEイオンとナトリウムと塩酸を取り、我々はLEバッファー100 mMのイオン強度を想定しています。 読者は、私たちのプロトコルのバッファイオン強度（および表1-2）は、常により大きいまたは10 mMに等しいであることに注意します。理論的にITPの物理学が10mMより低いイオン強度で適用されますが、1 mMのレベルに近い自然な汚染物質（水や大気中の二酸化炭素との反応から、例えばカルボン酸）は、しばしば低イオン強度バッファーの実際の使用を制限します。 我々はシグナル伝達機構は、このプロトコルで提示アッセイに限定されないことに注意してください。パート5で簡単に説明したように、高原モードで精製したゾーンは、ローカル伝導率の変化は、UV absorを介して検出することができます屈折のbance、温度、またはインデックスです。私たち自身のグループでは、我々は、未知の分析対象物質の高感度な検出と識別のための蛍光灯のキャリアアンフォライトを利用する方法を開発しました。ピークモードの実験では図5に示すように 、特定のプローブが焦点を当てた分析対象物にラベルを付けるために使用することができます。高い特異性で標的DNAまたはRNA分子を検出するために11,18を -彼らの標的配列にハイブリダイゼーションの際に蛍光オリゴヌクレオチドプローブ-一実施例では、我々は、分子ビーコンを使用しています。 ITPは、圧力駆動流とEOFによって引き起こされる分散に対して比較的ロバストである一方で、過度のEOFは、平均EOFの速度がITPの速度に等しくなるITPインタフェースの停滞につながる可能性があります。14このような強力なEOFは、特に高pHの条件で発生します。と低イオン強度。このような理由から、我々は、pH 8以下で、便利な場合100mMのオーダーのイオン強度を持つバッファを使用することをお勧めします。我々の経験では、PVPはホウケイ酸塩チップのEOFを抑制するための最も効果的なコーティング。しかし、そのふるいの特性から、PVPは、このようなゲノムDNAを中心としていくつかのアプリケーション用の適切でないかもしれません。実験では、PVPの添加が大幅に（それは集中していませんポイントに）ゲノムDNAの絶対モビリティを減らすことができます観察します。これらのケースでは、界面活性剤（例えば、トリトンX-100）と、組み合わせてSigmacoteとしてシラノールコーティングはまた、EOFを減らすのに有効であること10 <table border="1"><tbody><tr><td></td><td colspan="4"> TEアニオン（pKaは-1） </td></tr><tr><td> バッファ対イオン（PKA +1） </td><td> MES（6.10） </td><td> MOPS（7.20） </td><td> HEPES（7.50） </td><td> トリシン（8.15） </td></tr><tr><td>エタノールアミン（9.50） </td><td> 21.41 </td><td> 20.40 </td><td> 17.38 </td><td> 22.85 </td></tr><tr><td>トリス（8.08） </td><td> 21.00 </td><td> 19.23 &lt;/ TD&gt; <td> 15.84 </td><td> 17.56 </td></tr><tr><td>ビス - トリス（6.40） </td><td> 18.22 </td><td> 10.41 </td><td> 7.30 </td><td> 5.23 </td></tr><tr><td>ピリジン（5.18） </td><td> 1.01 </td><td> 3.72 </td><td> 2.42 </td><td> 1.48 </td></tr></tbody></table> 表1。 LEが100 mM HCl及び200mMの緩衝対イオンで ​​あるアニオン性のITPで調整された純粋な検体ゾーンの実効移動度の大きさ（×10 -9 M 2 / V / s）である。 <table border="1"><tbody><tr><td></td><td colspan="4"> TEの陽イオン（PKA +1） </td></tr><tr><td> バッファ対イオン（PKA -1） </td><td> エタノールアミン（9.50） </td><td> トリス（8.08） </td><td> ビス-トリス（6.40） </td><td> ピリジン（5.18） </td></tr><tr><td> MES（6.10） </td><td> 35.57 </td><td> 21.96 </td><td> 16.39 </td><td> 10.45 </td></tr><tr><td> MOPS（7.20） </td><td> 35.47 </td><td> 20.92 </td><td> 9.76 </td><td> 3.93 </td></tr><tr><td> HEPES（7.50） </td><td> 35.35 </td><td> 19.97 </td><td> 7.77 </td><td> 2.88 </td></tr><tr><td>トリシン（8.15） </td><td> 34.77 </td><td> 16.72 </td><td> 4.50 </td><td> 1.44 </td></tr></tbody></table> 表2。カチオン性ITPで調整された純粋な検体ゾーンの実効移動度の大きさ（×10 -9 M 2 / V / s）のLEは、100mM塩化ナトリウム、200 mMのバッファリングの対イオンで ​​ある。

<table border="1"><tbody><tr><td colspan="4"> 試薬 </td></tr><tr><td> 名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>蒸留水</td><td> GIBCO </td><td> 10977 </td><td>アーゼ/ DNaseフリー</td></tr><tr><td>クロロックス超</td><td>クロロックス</td><td> 02489CT </td><td></td></tr><tr><td>水酸化ナトリウム（NaOH） </td><td>マリンクロット</td><td> 7708 </td><td></td></tr><tr><td>塩酸（HCl） </td><td> EMDケミカルズ</td><td> HX0603-4 </td><td></td></tr><tr><td> Trizma塩基（トリス） </td><td> Sigma-Aldrich社</td><td> T6066 </td><td></td></tr><tr><td>ポリビニルピロリドン（PVP） </td><td>ポリサイエン株式会社</td><td> 06067 </td><td> MW100万</td></tr><tr><td> HEPES </td><td> Sigma-Aldrich社</td><td> H-4034 </td><td></td></tr><tr><td>のAlexa Fluor 488カルボン酸</td><td>インビトロジェン</td><td> A20000 </td><td></td></tr><tr><td>トリシン</td><td> Sigma-Aldrich社</td><td> T-9784 </td><td></td></tr><tr><td>ビス - トリス</td><td> Sigma-Aldrich社</td><td> B4429 </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> AM9260G </td><td></td></tr><tr><td>トリトンX-100 </td><td> Sigma-Aldrich社</td><td> X100 </td><td></td></tr><tr><td>リゾチーム</td><td> Sigma-Aldrich社</td><td> L6876 </td><td></td></tr><tr><td> SYBRグリーンII </td><td>インビトロジェン</td><td> S7564 </td><td></td></tr><tr><td>エタノールアミン</td><td> Sigma-Aldrich社</td><td> 411000 </td><td></td></tr><tr><td>ローダミン6G </td><td>アクロスオーガニック（ヘール、ベルギー） </td><td> CAS 989-38-8 </td><td></td></tr><tr><td> L-アミノ酸</td><td> Sigma-Aldrich社</td><td> LAA21 </td><td></td></tr><tr><td> 電源のSYBR Green RNA-に-C T 1ステップキット </td><td>アプライドバイオシステムズ</td><td> 4389986 </td><td></td></tr><tr> &lt;TD&gt; PCRプライマー</td><td>統合されたDNAの技術</td><td></td><td></td></tr><tr><td colspan="4"> 装置 </td></tr><tr><td> 名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ホウケイ酸塩マイクロ流体チップ</td><td>キャリパーライフサイエンス社</td><td> NS12A </td><td>プラスチックキャディーの有無にかかわらず未</td></tr><tr><td>真空ポンプ</td><td>ガスト</td><td> DOA-P104-AA </td><td></td></tr><tr><td>ソースメータ</td><td>ケースレー</td><td> 2410 </td><td>定電流および定電圧動作モード</td></tr><tr><td>倒立落射蛍光顕微鏡</td><td>オリンポス</td><td> IX70 </td><td>照明用水銀ランプ（オリンパス）やLED（Thorlabs）を使用して、 </td></tr><tr><td>フィルターキューブ</td><td>オメガ</td><td> XF115-2 </td><td>励起/発光：ブルー/グリーン</td></tr><tr><td>安全なロックマイクロ遠心チューブ</td><td>エッペンドルフ</td><td> 022363204 </td><td> 1.5 mLの容量</td></tr><tr><td>遠心分離</td><td>エッペンドルフ</td><td> 5417C </td><td></td></tr></tbody></table> 表3。特異的試薬および装置。

on-chip isotachophoresis for separation of ions and purification of nucleic acids

電技術があるため可動部分のない、シンプルでコンパクトなシステムでは、流体および電気泳動プロセスのさまざまな操作を実行する独自の能力のマイクロアプリケーションの主食です。等速電気泳動（ITP）は、イオン移動度に基づいて百万倍濃縮1,2、および効率的な分離と抽出を実現することができ、シンプルで非常に堅牢な電テクニックです。たとえば、我々は分離と非標識の高感度検出にITPのアプリケーションを実証した3ほとんど、またはまったくサンプルの準備4月8日とし、細胞培養、尿、血液などの複雑なマトリックスからの核酸の抽出と精製にイオン分子（例えば、毒素、DNA、リボソームRNA、miRNAを）。9月12日 ITPは、焦点と流体チャネル·システム内に印加される電界と2つのバッファを用いて分離を実現しています。アニオン性の検体については、主要な電解質（LE）バッファが選ばれているその陰イオンとしてPersat らによって詳細に説明し、後続の電解質（TE）バッファー（実効移動度はイオンの観測ドリフト速度を説明し、アカウントへのイオンの電離状態をとり、の陰イオンよりも高い効果的な電気泳動移動度を持っているUCH 13）。 TEとLE間のインタフェースを確立した後、電界は、LEイオンはTEイオンが占有する領域から離れて移動するように適用されます。先にTEイオンの中間の効果的なモビリティのレースのサンプルイオンがLEイオンを追い越すので、それらはLE-TEインタフェース（以下、 &quot;ITPインターフェース&quot;と呼ばれる）でピント合わせはできません。さらに、TEとLEは、ITPのインタフェースで急な電場勾配を確立し、それぞれ低域と高導電性の領域を形成します。彼らは焦点としてこのフィールドの勾配は、サンプル種をpreconcentrates。適切な他の非焦点を当てた種から焦点を当て、対象種の精製におけるTEとLEの結果の選択と、最終的には、分離サンプル種のd分離。 ここではITPの基礎となる物理的な原則を確認し、2つの動作標準モードについて説明します。 &quot;ピーク&quot;と &quot;高原&quot;モード。ピークモードでは、比較的サンプルイオンがITPインターフェイスで狭いピークが重なっ内で一緒に焦点希釈します。高原モードでは、より豊富なサンプルイオンが定常状態の濃度に達するとその実効移動度順に並べられ、隣接する台地のようなゾーンに分離する。ピークと高原モードでは、同じ基礎となる物理学の外に発生するが、最初の分析対象成分の濃度および/またはサンプルの蓄積のために割り当てられた時間の量によって区別異なる制度を表しています。 我々は、最初に詳細にモデルのピークモードの実験を記述し、E.からの核酸の抽出のためのピークモードアッセイを実証する大腸菌の細胞培養。我々は分離を可視化するために、非焦点トレーサー（NFT）種を使用する場所、高原モードアッセイを提示することによって、単位および結論アミノ酸の定量を形成しています。

Watch this Scientific Journal Video about On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids at JoVE.com

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

等速電気泳動（ITP）は、毒素の検出からサンプル調製に至るまでのアプリケーションで堅牢な電分離と濃縮技術です。小分子および細胞培養溶解液からの核酸の精製の分離と検出：我々はITPと2具体例のアプリケーションにこのテクニックを適用する方法論の物理的原理を確認します。

イオンの分離及び核酸の精製のためのオンチップ等速電気泳動

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

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Current and Resistance

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24.2K ビュー.  Stanford University. 等速電気泳動（ITP）は、毒素の検出からサンプル調製に至るまでのアプリケーションで堅牢な電分離と濃縮技術です。小分子および細胞培養溶解液からの核酸の精製の分離と検出：我々はITPと2具体例のアプリケーションにこのテクニックを適用する方法論の物理的原理を確認します。

ビデオ: On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

核酸、タンパク質と低分子のReagentless検出用電気化学DNAバイオセンサーの作製

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

生物工学

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

マルチアングル照明とピクセル超高解像度を採用しLensfreeオンチップ·トモグラフィー顕微鏡

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

オンチップ·血管内皮の炎症性表現型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

ドライ フィルムのレジストを用いた電気化学マイクロ流体バイオ プラットフォーム: デバイス作製、チップの試金の準備、およびシステム操作

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

作製と内皮下電気抵抗を直接定量化する統合型電極を用いたオンチップ器官システムの検証

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

キャピラリー配列で複数の核酸の視覚的検出

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

準備、精製、および脂肪酸含有リポソームを用いる

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

有機溶媒混合物中の複雑なナノ構造へのガンマ修飾ペプチド核酸の自己集合

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.