著者は、ドイツ研究振興協会 (DFG) 許可番号 UR 70/10-01 と UR 70/12-01 下でこの作業を部分的に資金調達のために感謝したいと思います。

1 マイクロ流体バイオ センサーを用いた DFR 技術の作製 <ol><li>ウェーハの PI の準備。。
	<ol><li>カット PI 基板 6 にウェーハをラウンドします。脱水焼くに約 1 時間の 120 の ° C のオーブンで PI ウェーハを置く</li>。
	</ol></li> <li>リフトオフ プロセス用フォトリソグラフィまず。
	<ol><li>3,000 rpm、2,000 rpm の加速時 30 の回転するスピン コーターをプログラム/s の場所スピンコーター PI ウエハーと真空を適用する、それを修正します。リフトオフ プロセスを可能に、レジスト 2 mL を分注、スピンコーター プログラムを開始します。スピンコーターからウェハを削除し、ソフト焼く 100 で 2 分間ホット プレート上の PI ウェーハ ° C</li>
		<li>肉眼で PI 基板上の電極をパターニングのため目的のマスクを調整し、400 mJ/cm 2 UV 光にそれを露出する (365 nm)。わずか 1 分の場所ボウルにウェハ軌道シェーカーでレジスト マッチング開発者でいっぱいそれを振る</li>
		<li>余分なレジストを除去した後は、濡れたベンチに脱イオン水 (純水) で PI ウエハを洗浄し、乾燥後、圧縮空気を使用してシャワーを使用します</li>。
	</ol></li> <li>プラチナ電極の形成のための蒸着。
	<ol><li>預金 200 標準物理蒸着法を使用してウェーハ 19 にプラチナの nm</li>。
		<li>は、ウェハをボウルに配置します。ボウルに一致する除去を追加し、すべての余分なプラチナが削除されるまで軌道シェーカーでウェハを少し振りながらリフトオフ レジストを除去します。1.2.3 のステップで説明したように PI ウェーハを乾燥をすすいで</li>。
	</ol></li> <li>2 番目写真平版ステップ: 断熱層を形成します。
	<li>それを脱水する 5 分の 120 ° C に予熱したオーブンで PI ウェーハを置く</li> <ol>
		500 rpm 時の回転の <li>プログラム最初ステップ スピン コーターの 5 s。30 の 2 番目のステップをプログラムに 4,000 回転, 700 rpm/s の加速を持つ s</li>
		<li>PI ウェーハ スピン コーターに置き、真空を適用する、それを修正します。スピンコーターのプログラムを開始する前に適切なエポキシ系フォトレジストの 4 mL を分注します</li>。
		<li>ソフト焼くホット プレート上 95 ° C で 3 分間コーティング ウェハ</li>。
		<li>は、それぞれアライメント マークと眼のレンズを使用してウェーハ上の絶縁層のマスクを調整します。100 mJ/cm 2 UV 光にウェーハを公開 (365 nm).</li>
		<li>露光後ベークの 95 で 2 分間予熱したホット プレート上のウェハを配置 ° C</li>
		<li>ボウルにウェハを置き、軌道のシェーカーに適して開発者 (例えば 1-メトキシ-2-プロピル-acetat SU 8 本の場合、2 分間) とレジストを開発</li>。
		<li>イソプロパノールでまず PI ウェーハを洗い流しし、すすいで 1.2.3 のステップで説明するよう、それを乾燥します</li>。
		<li>ハード焼く 150、3 時間オーブンでフォトレジスト ° C</li>
	</ol></li> <li>電極の洗浄プラズマ援用します。
	<ol><li>標準プラズマ装置酸素プラズマを使用して、フォトレジスト残留物を除去します。100% O 2 の流れ、250 sccm の割合 3 分の合計時間と 200 W 低周波電源を使用して、洗浄プログラムをスタート</li>
		<li>プラズマ室に PI ウェーハを置いて、ガラス蓋を使用して接地板にウェハを修正、プラズマ プロセスを開始します</li>。
	</ol></li> <li>銀・塩化銀沈着: オンチップ参照電極を作成する。
	紫外線に敏感な粘着テープによるウエハの <ol><li>Passivate プラチナ接触パッド。5 mm 幅と 11 cm 長いストライプに箔をカットし、銀と供託すべきではない部分を保護するウェハに結び付けます</li>。
		<li>銀蒸着のシリカフューム戸棚に超音波槽 (35 kHz) を入れて、お風呂に銀電解液 (100 g/L Ag、pH 12.5) のコンテナーを挿入します。ソニックのお風呂を部屋の温度と電源を 10% に設定します 。
		注意: 銀電解質溶液は毒性が高く、慎重に処理する必要があります。煙を吸入しない必要があります</li>。
		<li>は、参照電極の一括コンタクトを定電流源に接続します。カウンター電極、標準接続ケーブルを使用して現在のソースに、銀の電解質溶液に浸漬銀線を接続します</li>。
		<li>DC に-4.5 の電流密度に電流源を設定 mA/cm 2 の銀蒸着速度約 0.3 μ m/分のソニックのバスを開始させ 10 分・ ディ ・水ですすぎ、ウェーハが実行現在ソース</li>
		<li>参照電極の塩素処理、超音波浴に 0.1 M 塩化カリウム (KCl) 溶液の容器を挿入します。ウェハと定電流源による KCl 溶液中浸漬白金電極の一括接触を接続します</li>。
		<li>+0.6 の電流密度と dc 電流源を設定 mA/cm 2、約 0.075 μ m/分の沈着率の結果ソニックのバスを開始し、電流 7.5 分の実行</li>
		<li>水洗、乾燥、PI ウェーハ、1.2.3 のステップで説明したようします</li>。
		<li>UV 後 UV 感光性テープを削除 (365 nm) 300 mJ/cm 2 ・ リンス ドライに記載されているステップ 1.2.3 として再度、PI ウエハーの露光します</li>。
	</ol></li> <li>3 番目の写真平版ステップ: マイクロ チャネルを作成する使用して DFRs。
	<ol><li>は、ウエハース (20 × 20 cm 2) のようなサイズに必要な DFR レイヤーをカットしました。露出ユニットに必要なマスクを修正し、肉眼を使用してマスクに DFR のレイヤーを整列します。250 mJ/cm 2 UV 光でレジストを照らす (365 nm).</li>
		<li>は、フォトレジストの保護紙をはがすし、42 ° C とソニック パワーに予熱 (35 kHz) の標準的なソニックのバスを使用して 1% 炭酸ナトリウム (Na 2 CO 3) 溶液を約 2 分間 (構造) によってレジストを開発100%。反作用をすぐに停止し、軌道のシェーカーで 1% 塩酸風呂に 1 分のレジストをシェイクします。1.2.3 のステップで説明したように、DFR 各層を乾燥をすすいで 。
		注: 必要合計では、4 つの DFR 層 1.7 の手順に記載されているように処理する: 1 つのチャネル層、1 つのカバー層および (端曲げからバイオ センサーを防ぐため) 2 つの裏面レイヤーします</li>。
	</ol></li> <li>、DFR のラミネーション層 PI 基板上。
	<ol><li>は、透明オーバーヘッド箔に PI ウェーハを置き、標準の粘着テープを使用してそれを修正します。それぞれの配置構造を用いた顕微鏡下で PI ウェーハ上チャネル DFR 層を調整します</li>。
		<li>、ラミネート用標準圧延ラミネーターと予 100 ° c 上のロールおよび下部ロール 60 ° C 圧設定 3 バー、0.3 m/分の場所の前方速度、ラミネーターの真ん中に固定の DFR 層とウェハと開始します。民主は、ラミネーターがプッシュされます。180 ° でウェハを回転後の手順を繰り返します</li>。
		<li>バイオ センサーの曲げを防ぐため、次の手順 1.8.1-1.8.2 ウエハの上に 2 つの開発裏面層を積層します</li>。
	</ol></li> <li>疎水性停止バリアを採用・ チップを封止します。
	<ol><li>削除 DFR チャンネルのフロント側からの保護の層を置く

<ol><li>Spindel, S., Sapsford, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+optical+detection+platforms+for+multiplexed+detection+of+proteins+and+the+need+for+point-of-care+biosensors+for+clinical+use%5BTitle%5D)+AND+%22Sensors+%28Basel%29%22%5BJournal%5D)">Evaluation of optical detection platforms for multiplexed detection of proteins and the need for point-of-care biosensors for clinical use.</a> Sensors (Basel). 14 (12), 22313-22341 (2014).</li>
<li>Luppa, P. B., Bietenbeck, A., Beaudoin, C., Giannetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinically+relevant+analytical+techniques%2C+organizational+concepts+for+application+and+future+perspectives+of+point-of-care+testing%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Clinically relevant analytical techniques, organizational concepts for application and future perspectives of point-of-care testing.</a> Biotechnol Adv. 34 (3), 139-160 (2016).</li>
<li>Gauglitz, G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25014344">Point-of-Care Platforms.</a> Annu Rev Anal Chem. 7 (1), 297-315 (2014).</li>
<li>Jung, W., Han, J., Choi, J. W., Ahn, C. H. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0167931714004316">Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies.</a> Microelectron Eng. 132, 46-57 (2014).</li>
<li>Yager, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+diagnostic+technologies+for+global+public+health%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Microfluidic diagnostic technologies for global public health.</a> Nature. 442 (7101), 412-418 (2006).</li>
<li>Fu, E., Yager, P., Floriano, P. N., Christodoulides, N., McDevitt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perspective+on+diagnostics+for+global+health%5BTitle%5D)+AND+%22IEEE+Pulse%22%5BJournal%5D)">Perspective on diagnostics for global health.</a> IEEE Pulse. 2 (6), 40-50 (2011).</li>
<li>Yager, P., Domingo, G. J., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Point-of-care+diagnostics+for+global+health%5BTitle%5D)+AND+%22Ann+Rev+Biomed+Eng%22%5BJournal%5D)">Point-of-care diagnostics for global health.</a> Ann Rev Biomed Eng. 10, 107-144 (2008).</li>
<li>Dincer, C., Bruch, R., Kling, A., Dittrich, P. S., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+point-of-care+testing+-+xPOCT%5BTitle%5D)+AND+%22Trends+Biotechnol%22%5BJournal%5D)">Multiplexed point-of-care testing - xPOCT.</a> Trends Biotechnol. 35, (2017).</li>
<li>Horak, J., Dincer, C., Bakirci, H., Urban, G. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0925400513012057">A disposable dry film photoresist-based microcapillary immunosensor chip for rapid detection of Epstein-Barr virus infection.</a> Sens Actuators B Chem. 191, 813-820 (2014).</li>
<li>Method for the fabrication of a &#34;lab on chip&#34; from photoresist material for medical diagnostic applications. US patent. , 7,691,623 (2010).</li>
<li>Kling, A., Dincer, C., Armbrecht, L., Horak, J., Kieninger, J., Urban, G. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1877705815024571">Electrochemical microfluidic platform for simultaneous multi-analyte detection.</a> Procedia Eng. 120, 916-919 (2015).</li>
<li>Armbrecht, L., Dincer, C., Kling, A., Horak, J., Kieninger, J., Urban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-assembled+magnetic+bead+chains+for+sensitivity+enhancement+of+microfluidic+electrochemical+biosensor+platforms%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms.</a> Lab Chip. 15, 4314-4321 (2015).</li>
<li>Dincer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designed+miniaturization+of+microfluidic+biosensor+platforms+using+the+stop-flow+technique%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Designed miniaturization of microfluidic biosensor platforms using the stop-flow technique.</a> Analyst. 141, 6073-6079 (2016).</li>
<li>Weltin, A., Kieninger, J., Enderle, B., Gellner, A. K., Fritsch, B., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymer-based%2C+flexible+glutamate+and+lactate+microsensors+for+in+vivo+applications%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">Polymer-based, flexible glutamate and lactate microsensors for in vivo applications.</a> Biosens Bioelectron. 61, 192-199 (2014).</li>
<li>Kling, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multianalyte+Antibiotic+Detection+on+an+Electrochemical+Microfluidic+Platform%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Multianalyte Antibiotic Detection on an Electrochemical Microfluidic Platform.</a> Anal Chem. 88 (20), 10036-10043 (2016).</li>
<li>Bruch, R., et al. Clinical on-site monitoring of ß-lactam antibiotics for a personalized antibiotherapy. Sci Rep. , (2017).</li>
<li>Horak, J., Dincer, C., Qelibari, E., Bakirci, H., Urban, G. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0925400514015482">Polymer-modified microfluidic immunochip for enhanced electrochemical detection of troponin i.</a> Sens Actuators B Chem. 209, 478-485 (2015).</li>
<li>Horak, J., Dincer, C., Bakirci, H., Urban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive%2C+rapid+and+quantitative+detection+of+substance+P+in+serum+samples+using+an+integrated+microfluidic+immunochip%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">Sensitive, rapid and quantitative detection of substance P in serum samples using an integrated microfluidic immunochip.</a> Biosens Bioelectron. 58, 186-192 (2014).</li>
<li>Mattox, D. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMattox%2C+DM+%22Handbook+of+Physical+Vapor+Deposition+%28PVD%29+Processing%22">Handbook of Physical Vapor Deposition (PVD) Processing</a>. , Elsevier Inc. (2010).</li>
</ol>

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マイクロ流体電気化学バイオ センサーの作製はここで説明したプロトコルにより、生体分子の検出のための低コスト、コンパクト、簡単に使用できるプラットフォームの開発。バイオ センサーでその後使用されるアッセイ、に応じていくつかの異なるマーカーを検出できます。これは、プラットフォームは、非常に汎用性とポイント ・ オブ ・ ケア アプリケーションに標準的な診断テスト...

過去二十年以上診断アプリケーションは世界の公衆衛生の開発に関する詳細な研究の基礎になっています。伝統的に、病気の検出実験室の診断ツールを使用します。にもかかわらず、彼らはまだ病気の診断に重要な役割を再生、ポイント ・ オブ ・ ケア ・ テスト (POCT) 患者の近くを実行または患者自身によって近年ますます一般的になっています。急性心筋梗塞や糖尿病のモニタリングなど、すぐに治療を必要とするような場合に特に臨床知見の迅速な確認が欠かせません。したがって、POCT 機器非専門家を運営することができ、同時に短時間1,2,3,4 で正確な体外診断テストを実行することが可能である必要があります。.
顕著な改善は、POCT の分野ですでに達成されています。しかし、まだ5,6,7、8を克服するために多くの課題があります。POCT プラットフォーム市場を正常に起動して実験室の診断と競合するデバイスは厳密に次の要件を満たす必要があります: (i) は研究所と一致している正確で定量的なテスト結果を提供します。調査結果;(ii) 患者の即時の処置を有効にする時間の短いサンプル結果があります。(iii) 未熟な個人によって動作する場合でも、シンプルで簡単な処理を機能し、最小のユーザー介入を必要とします。(iv) 単一用途のために設計された低コストのセンサー ユニットを構成します。さらに、無料の設備診断、資源の乏しい環境3,4,6を中心に、有利です。
これらの厳しい要件のため電気化学検出 (例えば、血ブドウ糖テスト ストリップ) と側方流動免疫測定 (例えば、ホーム妊娠検査) に基づいてのみ 2 つの POCT システム開始されている正常に市場にそうこれまで。しかし、両方のシステムはパフォーマンスの低下などの欠点に苦しむ (不正確なテスト結果をすなわち、血ブドウ糖の監視と横の流れの試金は質的 (正または負) の測定結果を提供するだけ)4、 6。従来の POCT システムのこれらの欠点は、介護4、5の時点で高速、低コスト、および定量的な検出を提供する新しい技術の探索の需要の増加につながっています。
DFR 技術 POCT 機器が直面する課題を満たすためには、使い捨て・低コストのバイオ センサー9,10、11,12,の作製の最近採用されています13,14. DFRs PDMS や SU-8 などのソフトと液体のリソグラフィ材料に比べて多くの利点を提示: (i) 発行しております様々 な組成と厚さ (から数ミリ、数ミクロン);(ii) 各種材料への接着を容易に非常に大まかな表面領域があります。(iii) 機能優れた膜厚均一性;(iv) 量産の安易な安い、高スループットの作製を提供します。(v) ではさみ; シンプルなペアのような様々 な低コストのツールで簡単にカット(vi) 互いの上に複数の DFR 層を積層することによりマイクロ流路などの三次元構造の作成を可能にします。
その一方で、DFRs の一般マスクと保護箔、さらに光を可能にするため DFR の増加の距離の膜厚によって主に引き起こされる液体のフォトレジストと比較して解像度が比較的低いがあります。散乱。まだ、統合されたマイクロ流体バイオ センサーの製造、DFRs が低コストで量産に最適です。
したがって、私たちはこの存在は DFR に基づく電気化学マイクロ流体バイオ センサーの作製と機能します。詳細なプロトコルでは、バイオ センサー プラットフォームの各生産段階、オンチップの固定化 DNA ベースのモデル分析とストップ フロー法を用いたその電気化学的読み出しをについて説明します。この普遍的なプラットフォームは、さまざまな分析技術 (例えばゲノミクス、cellomics、およびプロテオミクス) またはアッセイ フォーマット (例えば、競争力のある、サンドイッチ、または直通) 生体分子の多数の種類の検出を有効。 にします。このような DFR プラットフォームに基づいて、私たちのグループ以前実証抗生物質13,15,16 (テトラサイクリンを含む、様々 な検体の迅速・高感度定量プリスチナマイシンと β-ラクタム系抗生物質)、トロポニン私17、およびサブスタンス P18。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pyralux</td><td>DuPont</td><td>AP8525R</td><td>Used as polyimide substrate</td></tr><tr><td>MA-N 1420</td><td>Micro Resist Technology</td><td>MA-N1420</td><td>Lift-off resit to define the platinum depostion</td></tr><tr><td>Ma-D 533s</td><td>Micro Resist Technology</td><td>MaD533S</td><td>Developer for MA-N1420</td></tr><tr><td>Platinum</td><td>-</td><td>-</td><td>Electrode and contact pad material</td></tr><tr><td>Ma-R 404s</td><td>Micro Resist Technology</td><td>MaR404S</td><td>Remover for MA-N1420</td></tr><tr><td>SU-8 3005</td><td>MicroChem Corp.</td><td>SU-8-3005</td><td>Photoresist to define the electrode area and as insulation</td></tr><tr><td>1-methoxy-2-propanol acetate</td><td>Sigma-Aldrich</td><td>108-65-6</td><td>Developer for SU-8 3005</td></tr><tr><td>2-Propanol</td><td>VWR</td><td>8.18766.2500</td><td>Removing of the SU-8 developer</td></tr><tr><td>1020R</td><td>Ultron Systems Inc.</td><td>1020R</td><td>UV sensitive adhesive tape for protection of contact pads</td></tr><tr><td>Arguna S</td><td>Degussa</td><td>1935</td><td>For Silver depostion on reference electrode</td></tr><tr><td>KCl</td><td>Methrom</td><td>62308.020</td><td>For chloridation of the silver reference electrode</td></tr><tr><td>Pyralux</td><td>DuPont</td><td>PC1025</td><td>Dry film photoresist</td></tr><tr><td>Sodium carbonat</td><td>Fluka</td><td>71352</td><td>Developer for Pyralux PC1025</td></tr><tr><td>Hydrogen chloride</td><td>Sigma-Aldrich</td><td>30720</td><td>To top the development of the DFR</td></tr><tr><td>Teflon AF 1600</td><td>DuPont</td><td>AF1600</td><td>For employing the stopping barrier</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PA104</td><td>Mega Electronics</td><td>-</td><td>Bubble etch tank</td></tr><tr><td>FED 53</td><td>Binder</td><td>9010-0018</td><td>Oven</td></tr><tr><td>SPIN150</td><td>APT</td><td>-</td><td>Spin coater</td></tr><tr><td>Pr&amp;auml;zitherm</td><td>Harry Gestigkeit GmbH</td><td>PZ 28-2</td><td>Hot plate</td></tr><tr><td>Hellas</td><td>Bungard Elektronik</td><td>40000</td><td>Exposure unit</td></tr><tr><td>Tetra30-LF-PC</td><td>Diener</td><td>-</td><td>Plasma unit</td></tr><tr><td>Univex 500</td><td>Leybold</td><td>-</td><td>Physical vapor deposition unit</td></tr><tr><td>Shaker S4</td><td>ELMI</td><td>-</td><td>Orbital shaker</td></tr><tr><td>Sonorex Super 10 P</td><td>Bandelin</td><td>783</td><td>Sonic bath</td></tr><tr><td>6221 DC and AC</td><td>Keithley</td><td>-</td><td>Current source</td></tr><tr><td>HRL 350</td><td>Ozatec</td><td>-</td><td>Laminator unit</td></tr><tr><td>Vaccum pen</td><td>EFD</td><td>-</td><td>Vacuum pen</td></tr></tbody></table>

dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation

近年、バイオ マーカー診断ポイント ・ オブ ・ ケア診断にとって、人間の病気の診断のために欠かせないツールとなった。使いやすい、低コストのセンサー プラットフォームは、定量的かつ具体的にはさまざまな種類の分析 (例えば、バイオ マーカー、ホルモンや薬) を測定するが望ましい。このため、ドライ フィルムのレジスト技術 - 安価な安易な、高速加工 - を有効にするは、ここで紹介するマイクロ流体バイオ センサーを製造する使用されました。その後使用される生物検定によって汎用性の高いプラットフォームは、生体分子の様々 なタイプの検出が可能です。デバイスの作製、白金電極のみクリーン ルーム工程で柔軟性の高いポリイミド (PI) 箔で構成されています。PI 箔はエポキシ系フォトレジスト絶縁電極用基板として提供しています。マイクロ流路がその後開発と PI ウエハの上にドライ フィルムのレジスト (DFR) 箔の積層によって生成されます。チャネルの疎水性停止バリアを使用して、チャネルは、2 つの特定の領域に分かれています: 酵素試金のアンペロ メトリック信号の読み出しのための電気化学的測定セル固定セクション。
チャネル表面への生体分子の吸着によるオンチップ生物検定の固定化が実行されます。グルコース酸化酵素酵素は、電気化学信号のトランスデューサーとして使用されます。基板、グルコースの存在下でこれがプラチナの作用電極で検出された過酸化水素が生成されます。ストップ フロー法は、迅速な検出と信号増幅を得るに適用されます。異なる生体分子は、病気のまたは監視、パーソナライズされた療法を促進する治療薬に関しては、さまざまな種類の指標を与えて導入マイクロ流体システムによる定量的測定できます。

設計とマイクロ流体バイオ センサー プラットフォームの試作:
ウエハー レベルで複数 DFR 層を用いた標準写真平版の技術によりマイクロ流体バイオ センサー チップの作製を実現します。この製作方法は、マイクロ流路の形成、プラチナ パターン PI 基板 DFRs の先進層の積層に依存します。作製手順を描いた短い概要は、<str...

Watch this Scientific Journal Video about Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation at JoVE.com

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

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

マイクロ流体バイオ センサー プラットフォームは、設計された、様々 な検体の迅速・高感度な定量化のため低コスト ドライ フィルム現像液の技術を用いて作製しました。この単回使用システムは、ストップ フロー法による上チップ-固定化酵素試金の電気化学的読み出しができます。

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

dry-film-photoresist-based-electrochemical-microfluidic-biosensor

Research

JoVE Journal

Bioengineering

11.7K ビュー.  University of Freiburg. マイクロ流体バイオ センサー プラットフォームは、設計された、様々 な検体の迅速・高感度な定量化のため低コスト ドライ フィルム現像液の技術を用いて作製しました。この単回使用システムは、ストップ フロー法による上チップ-固定化酵素試金の電気化学的読み出しができます。

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

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

可逆小分子またはバイオセンサー応用のためのマルチコンポーネント構造を固定化するために、マイクロ流体オンチップキャプチャ - 環化反応

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

化学

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

生物工学

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

用絹フィルム培養システム in vitroで分析とマテリアルデザイン

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

単一細胞の多彩な化学分析マイクロ流体チップ

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

ラベルフリーDNAハイブリダイゼーション分析のためのマイクロ流体ベースの電気化学バイオチップ

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

自動プロテオーム処理のためのデジタルマイクロ流体

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

生物学

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

マイクロリアクター及び、電磁作動に水滴が酵素として使用

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

粒子の空間追跡のための多重化された電子の検出とマイクロ流体プラットフォーム

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

酵素結合免疫吸着アッセイ用エレクトロウェッティングベースのデジタルマイクロ流体プラットフォーム

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

メカノノードポアセンシング:マルチパラメータシングルセル粘弾性測定のための迅速でラベルフリーのプラットフォーム

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

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

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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

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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