サンディア国立研究所は、ユナイテッドが契約DE-AC04-94AL85000下でエネルギー省の国家核安全保障管理局をすればのサンディア国立研究所株式会社、ロッキードマーチン社が運営するマルチプログラムの研究室です。著者らは、サンディア国立研究所 &quot;研究所からの資金認める研究開発（LDRD）プログラムを監督。

1。マイクロニードル作製<ol><li>三次元モデリングソフトウェアSolidworksで（Dassault SystemesのSA、ツィー、フランス）を使用して、ピラミッド状の中空マイクロニードルアレイを設計する（ 図1）。3 月5日 </li><li> Magicsは、RP 13ソフトウェア（マテリアライズNV、ルーベン、ベルギー）を使用してマイクロニードルアレイのための支持構造を設計します。支持構造は、樹脂製造時にデバイスからドレインすることができ、マイクロニードルが構築されている基盤を提供しています。例の支持構造は、 図1に示されています。 </li><li>リンクされているサポートとマイクロニードルアレイのファイルは、製造プロセスを制御するPerfactory RPソフトウェア（EnvisionTEC GmbHは、Gladbeck、ドイツ）にアップロードされます。このソフトウェアパッケージ内に、作製するマイクロニードルアレイの数を選択し、製造プレート上のデバイスの配置を決定します。 </li><li> Perfactory迅速なPRは180 mWで紫外線モードでキャリブレーションを実行します。ototyping製造システムとエネルギーのずれを確認して±2 mWの範囲内である。 </li><li>製作が完了したら、15分間イソプロパノールでベースプレートからマイクロニードルアレイを削除して開発しています。圧縮空気を持つ配列を乾燥させ、完全な重合を確実にするためにOtoflash Postcuringシステム（EnvisionTEC社、Gladbeck、ドイツ）で50秒間室温でマイクロニードルを治す。 </li><li>顕微鏡を介してマイクロニードルの製造を検証し、各マイクロニードルの穴は​​中空と障害物がないことを確認してください。完全に製作されたマイクロニードルは、 図2に示されています。 </li></ol> 2。カーボンペースト電極アレイの作製<ol><li>穴をカットしてあったフラットフレキシブルケーブル（21039から0249）で、基礎となる個々にアドレス指定接続する銅線を露出する60 Wモデル6.75 CO 2ラスター/ベクトルレーザーシステム（ユニバーサルレーザーシステムズ社、スコッツデール、アリゾナ州）を使用して、商業的供給源（Moから得られたlexのコネクタ（株）、リール、イリノイ州）（ 図3（AとB））。適切にレーザーアブレーションプレート上に配置する治具でフラットフレキシブルケーブルを配置します。フレキシブルケーブルの絶縁部に500μmの直径の空洞を作成するラスタアプローチを使用します。アブレーション用のパターンはCorelDRAWの作成（コーレル、オタワ、オンタリオ州）とレーザーシステムに送信されます。 </li><li> 40 psiでエアブラシそのスプレーのアセトンで変更されたフラットフレキシブルケーブルをきれいにします。イソプロパノール、脱イオン水で洗浄して、それらを洗浄終了します。ない絶縁膜が露出した銅ストリップの上に残っていないことを顕微​​鏡下で確認してください。 </li><li>次のステップでは、カーボンペーストの梱包の保持キャビティを作成することです。メリネックステープ（粘着アクリル粘着剤を片面にコーティングされた0.002 &quot;厚さ）は2分間の適切な接続を確保するためにアブレーション電極ストリップの上に配向し、電極ストリップと同じパターンでアブレーション、3000 psiで圧縮されています。でこのCASEは、空洞の直径は750μmである。 </li><li>メリネックステープ（粘着アクリル系粘着剤の2つの両面に塗布0.004 &quot;厚さ）の追加の層は、その後、片面粘着テープと同じパターンでアブレーションされており、債券に整列した後にカーボンペースト電極アレイのマイクロニードルアレイに使用されます。 </li></ol> 3。機能性カーボンペーストの合成と電極キャビティのパッキング<ol><li>グルコース敏感カーボンペーストは、以前のレシピのオフに基づいており、均一な混合物が得られるまでグルコースオキシダーゼおよびポリ（エチレンイミン）の2.2 mgの10mgを混合することによって得られる。この混合物に16、ロジウム60 mgのカーボン粉末上に（ 5パーセントの負荷）が追加されます。 40 mgのミネラルオイルを添加し、続いて混合される。ペーストを調製後1週間まで使用されます。ペースト°C使用時まで4℃で保存されています。 </li><li> pH感受性カーボンペーストは、混合30％（w / w）の鉱物油と70％（w / w）の黒鉛pによって得られたowder。セクション3.4で説明したように電極の空洞に貼り詰める。 、17の10mMファーストブルーRRジアゾニウム塩（4 -ベンゾイルアミノ-2,5 - dimethoxybenzenediazonium塩化ヘミ（塩化亜鉛）塩）0.5 Mリン酸の溶液を作るためにパックされたペースト電極上にこの溶液20μl滴を置きます自発的にファーストブルーPRのジアゾニウム塩を化学吸着すること30分。緩衝液または脱イオン水使用しないで脱イオン水や店舗で洗い流してください。 </li><li>乳酸敏感カーボンペーストは、以前のレシピをベースにしていますと、炭素粉末と乳酸オキシダーゼ2.5mgのにロジウムの2.5 mgを混合することによって得られる、5回転のために超音波5分、ボルテックスの5分の間で交互に18。 </li><li>準備されたフラットフレキシブルケーブルに変更されたペーストの梱包は、電極の空洞を介してそれぞれのペーストを適用することによって達成されます。こてとパックtとしてプラスチックの薄い部分を（例えば、プラスチック重船のエッジ）を使用して、滑らかな表面が得られるまで、彼は貼り付けます。過剰なペーストが削除されるまで、2番目のきれいな秤量ボートで繰り返します。脱イオン水で洗い流して下さい。カーボンペーストの充填キャビティ、およびマイクロニードルの統合を（セクション2で説明した、3）を作成するレーザーアブレーションを示す模式図を図3に示されています。 </li></ol> 4。検出とセンサの校正<ol><li>乳酸の検出は、-0.15 Vでセンサのクロノアンペロメトリ応答を測定し、0.1 Mリン酸緩衝液（pH = 7.5）で15秒後に電流を記録することによって達成されます。 図4（）は乳酸を検出するための電極触媒反応の模式図が含まれています。 </li><li>グルコースの検出は-0.05 Vでセンサのクロノアンペロメトリ応答を測定し、0.1 Mリン酸緩衝液（pH7.0）で15秒後に電流を記録することによって、同様の方法で実行されています。 図4（b）のための電極触媒反応の模式図が含まれていますDグルコースのetection。 </li><li> pHは、100 mVの/ sで-0.7 Vから0.8 Vにサイクリックボルタンメトリーのスキャンを実行すると酸化ピーク電位の位置を記録することによって監視されています。 pHの検出のための酸化還元反応の模式図を図5に示されています。 </li><li>グルコースと乳酸センサーのキャリブレーション曲線は、それぞれの検体の連続添加することによって作成することができます。セクション5.1と5.2で説明したようにクロノアンペロメトリ測定は、各検体の添加後、実行している。また、固定電位クロノアンペロメトリ測定は、現在の安定化のため各検体の添加の間に十分な時間を（〜10から100秒）ながら攪拌下に行うことができます。 </li><li> pHの検量線は1.0 pH単位の増分で5〜8に知られているpH値は、一連の上に酸化ピーク電位の位置を測定し、セクション5.3で説明したようにサイクリックボルタモグラムを記録することによって作成することができます。 </li></ol> 5。代表的な再sults 修飾カーボンペースト充填ニードルを静止溶液中でのクロノアンペロメトリ曲線を（例えば、グルコースの検出または乳酸を検出するための）取得する場合、電流はすぐにそれぞれの検出電位の印加により減少します。それは最終的に定常状態の値に減衰します。代表的な結果を図6に示されているが、この結果は、乳酸マイクロニードルで乳酸や記録の2mMの追加から得られた。ソリューションは、簡単にそれぞれの乳酸を添加した後攪拌しなければなりません。 15秒後に電流が乳酸の濃度を増加させる時に上昇し、現在の応答は、未知の溶液中での乳酸の濃度を決定するために使用することができます。また、継続的な監視は、増加するグルコース濃度（ 図5）で解決策を示すように撹拌した溶液（または流れる溶液中）で使用することができます。再び、時の電流の増加は、tを増加させる彼のグルコース濃度が未知のソリューションへのグルコース応答を標準化するために使用することができます。十分な時間は、溶液を安定させるために、各スパイクの後に許可する必要があります。 0.1 Mリン酸緩衝液のpHに敏感なマイクロニードルでのサイクリックボルタモグラムを図6の1のpH単位の増分で5から8までの4つの異なるpH溶液を介して表示されます。 pHの上昇による酸化ピーク電位のシフトが、この現象は、pH値の指標として使用されています。 <img alt="図1" src="/files/ftp_upload/4067/4067fig1.jpg" /> SolidWorksで作成したマイクロニードルアレイ（）のSTLファイルのサポート体制（B）を示し、印刷画面の図は、1。画像。 <img alt="図2" src="/files/ftp_upload/4067/4067fig2.jpg" /> 図2マイクロニードルアレイ（A）と、この配列内の単一のマイクロニードル（B）の走査型電子顕微鏡写真。 図3：フラットフレキシブルケーブルアセンブリの模式図。手順は、フラットフレキシブルケーブル（）を変更（B）パターン化され、円を切除、カーボンペースト（C）で満たされたと同様に、第二アブレーションメリネックス層を追加し、交配された当初はアブレーションメリネックス層を追加、含まれていマイクロニードルアレイ（D）が。 <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図4" src="/files/ftp_upload/4067/4067fig4.jpg" /> 図4。0.1 Mリン酸緩衝液（pH = 7.5）で-0.15 Vで15秒クロノアンペロメトリスキャンと乳酸敏感なペーストのキャリブレーション。乳酸の2mM加えて、現在対応しての各増加。 <img alt="図5" src="/files/ftp_upload/4067/4067fig5.jpg" /> 図5。 </s応答内の各減少は、グルコースの2mMの加算に対応する-0.05 Vの時クロノアンペロメトリスキャンを実行するグ​​ルコース感受性ペースト&gt;キャリブレーションをtrong。 0.1 Mリン酸緩衝液（pH = 7.0）は、キャリブレーション中に攪拌した。外部Ag / AgCl電極とPt基準と対向電極は、本研究で使用されていた。 <img alt="図6" src="/files/ftp_upload/4067/4067fig6.jpg" /> 図6 1。pHの単位の増分（緑青= pHが8.0、緑= pHは7.0、紫= pHが6.0、赤= pH 5.0）でのpH 5から8以上0.1 Mリン酸緩衝液に貼り付けpH感受性炭素のサイクリックボルタモグラム（CV）。第五CVはAg / AgCl参照とPt線カウンター電極対分析のために使用されていました。

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利害の衝突が宣言されません。

このニードルベースの​​センサの設計の複数の側面は、デバイス製造の前に考えられた。リアルタイムの検出のため、このセンサーを使用するために、センサの応答時間は、低でなければなりません。このプロトコルでは、各テストセンサーが15秒未満の応答時間を示した。このプロトコルで使用されているペーストは、電極の応答を妨げる可能性が電気の生体分子を含む生体内環?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>フラットフレキシブルケーブル</td><td>モレックス</td><td> 3302/10SF </td></tr><tr><td> 0.003 &quot;サイド両面テープ</td><td>メリネックス</td><td></td></tr><tr><td> 0.004 &quot;両面テープ</td><td>メリネックス</td><td></td></tr><tr><td>乳酸オキシダーゼ</td><td>シグマ</td><td> L0638 </td></tr><tr><td>ブドウ糖酸化酵素</td><td>シグマ</td><td> G7141 </td></tr><tr><td>炭素上のロジウム</td><td>シグマ</td><td> 206164 </td></tr><tr><td>黒鉛粉末</td><td>シグマ</td><td> 385031000 </td></tr><tr><td>ポリ（エチレンイミン） </td><td>アクロス</td><td> 178570010 </td></tr><tr><td>鉱油</td><td>シグマ</td><td> M5904 </td></tr><tr><td>グルコース</td><td>シグマ&lt;/ TD&gt; <td> G8270 </td></tr><tr><td>乳酸塩</td><td>シグマ</td><td> L1750 </td></tr><tr><td>ファーストブルーRR塩</td><td>シグマ</td><td> F0500 </td></tr><tr><td>電子シェル300 </td><td> EnvisionTEC </td><td></td></tr><tr><td>電子シェル200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ag / AgCl参照電極</td><td>バシ</td><td> MF-2052 </td></tr><tr><td> Pt線</td><td>バシ</td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> Perfactory RP </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ottoflash Postcuringシステム</td><td> EnvisionTEC </td><td></td></tr><tr><td>リン酸</td><td>フィッシャー</td><td> A366-4 </td></tr><tr><td> 60Wモデル6.75 CO 2ラスター/ベクトルレーザーシステム</td><td> UniverSALレーザーシステム</td><td> PLS6.75 </td></tr><tr><td> CorelDrawに</td><td>コー​​レル</td><td></td></tr><tr><td> SolidWorksの</td><td>ダッソー·システムズ</td><td> 2009 </td></tr><tr><td>魔法RP13 </td><td>マテリアライズ</td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

生体関連分子の迅速分析のための低侵襲多重監視システムの開発は、慢性疾患から彼らの即時の生理学的状態の容易な評価をしている個体を提供することができます。さらに、複雑な、多因子の医学的状態の分析のための研究ツールとして役立つかもしれません。実現するなどmultianalyteセンサーのためには、ユーザーへの痛みや害がなく発生する必要があります間質液のサンプリング、低侵襲である必要があり、分析が迅速なだけでなく、選択的でなければなりません。 最初は痛みの薬物送達のために開発され、マイクロニードルが皮膚を通してワクチンや薬理学的薬剤（例えば、インスリン）を提供するために使用されている1-2これらのデバイスは格子間空間をアクセスするので、微小電極と統合されているマイクロニードルは、経皮として使用することができます電気化学センサー。グルコース、グルタミン酸、乳酸の選択的検出、Hydrogen過酸化水素、アスコルビン酸が伝達要素として、炭素繊維、変性されたカーボンペースト、白金コーティング、ポリマーマイクロニードルと統合されたニードル電極デバイスを使用して実証されている。3-7,8 このニードルセンサー技術は、複数の分析物のin situ同時検出のための小説と洗練された分析的なアプローチを可能にしました。多重化は、特に迅速かつ低侵襲な方法で特徴づけることが困難な複雑な微小環境を監視する可能性を提供しています。たとえば、この技術は、グルコース、乳酸およびpHの細胞外レベルの同時モニタリング、疾患状態7,10-14（例えば、癌の増殖）および運動誘発性アシドーシスの重要な代謝指標である9のために利用される可能性があります。15

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

この資料では、詳細多重ニードルベースの​​センサの構築を。デバイスは、迅速かつ選択的に複数の分析物のin situ電気化学的サンプリングと分析のために開発されています。臨床医学と生物医学研究は、これらのマイクロニードルベースの​​センサに使用する私たちは想像する。

多重経皮電気化学センシングのための中空マイクロニードルベースの​​センサ

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

Research

JoVE Journal

Bioengineering

14.2K ビュー.  University of North Carolina and North Carolina State University. この資料では、詳細多重ニードルベースの​​センサの構築を。デバイスは、迅速かつ選択的に複数の分析物のin situ電気化学的サンプリングと分析のために開発されています。臨床医学と生物医学研究は、これらのマイクロニードルベースの​​センサに使用する私たちは想像する。

ビデオ: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

干渉反射イメージングセンサー（IRIS）を用いた生体分子の検出

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

生物工学

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

磁気ピンセットを用いた多重化単分子フォースタンパク質分解測定

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

電気化学センサを用いた細菌検出＆識別

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

有機電気化学トランジスタにバリア組織破壊の検出

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

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 ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

溶剤アシスト脂質二重（SALB）法による生体膜の作製

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

用中空糸バイオリアクター<em&gt;インビボ</em&gt;哺乳動物の組織培養様

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

マイクロカプセル化アプリケーション用の抽出プラントベースのカプセル

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

架橋反応性無料、スポッター無料多重サンドイッチ免疫測定用チップをスナップします。

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

食品由来病原体を検出する電気化学的 DNA のバイオ センサーの開発

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

多重経皮電気化学センシングのための中空マイクロニードルベースの​​センサ

多重経皮電気化学センシングのための中空マイクロニードルベースのセンサ