本研究では、アレルギーと国立感染症研究所からと小児泌尿器科研究の卓越性のためのウェンディとケンルビー基金協力協定賞AI075565（DAHに）によってサポートされていました。 BMCは、小児泌尿器科でジュディスとロバート·ウィンストンの椅子です。

1。電気化学センサーの機能化<ol><li> 300μMの1,6 - ヘキサン（HDT）、10mMのトリス-HCl、pH8.0、0.3 MのNaCl、1mMのEDTA、10分間、室温で暗所でインキュベートにおいて0.05​​μMの濃度でチオール化捕捉プローブを準備。 HDTを有するチオール化捕獲プローブのインキュベーションは、より一貫性のある結果をもたらし、捕捉プローブのチオール基が低減されることを保証する。 </li><li>水分および/または粒子を除去するために5秒間裸金16センサアレイチップ（複数可）に窒素気流を適用する。 </li><li> HDT-チオール捕捉プローブの6μlのセンサアレイのすべての16のセンサーの作用電極に混合し、4℃で一晩カバーペトリ皿にセンサーチップ（s）を保存し適用します。チオール捕捉プローブは、裸の金電極に直接結合し、HDTは、捕捉プローブの過剰充填防止し、それらの目標とのハイブリダイゼーションを促進し、拡張コンフォメーションを保つように作用する。 </li><li>ザ翌日、5秒間、窒素気流下で2〜3秒で乾燥し、脱イオンH 2 Oとセンサチップを洗う。 </li><li> 10mMのトリス-HCl、pH8.0、0.3 MのNaCl、1mMのEDTA、1mMの全てのセンサ16と50分間インキュベート作用電極に6 - メルカプト-1 - ヘキサノール（MCH）6μlのを適用する。これ以降のすべてのセンサチップインキュベーションを室温で覆われたペトリ皿で​​行われる。 MCHは、チオール捕捉プローブまたはHDTは、電極表面上に存在しない任意のギャップを埋める、ブロッキング剤として機能します。 </li></ol> 2。試料調製<ol><li> 5分間16,000 xgで遠心チューブと遠心分離機に成長の対数期（≈0.1 OD 600）で細菌培養液1mlを転送します。培養上清を取り除きます。細菌ペレットを直ちに処理または-80°C、後で使用するために保存することができる。 </li><li>徹底的にボットにピペットチップを適用することにより、1MのNaOH10μlに細菌ペレットを再懸濁マイクロチューブのトムと数回ピペッティング。 5分間室温で懸濁液をインキュベートする。 </li><li>ウシ血清アルブミン（BSA）およびフルオレセイン変性検出プローブの0.25mMの2.5％を含有し、pHが7.2、1 Mリン酸緩衝液50μlを添加して細菌溶解物を中和する。室温で10分間中和された溶解物をインキュベートする。フルオレセイン変性検出プローブは、細菌リボソームRNA標的分子とハイブリダイズする。 </li></ol> 3。電気化学センサーアッセイ<ol><li> 5秒のために、窒素気流下2-3秒、乾燥のために脱イオンH 2 OとのセンサーチップからMCHを洗ってください。 </li><li> 14センサーのそれぞれの作用電極に中和細菌溶解4μlのを適用し、15分間インキュベートする。標的検出プローブ複合体は、固定されたチオール化捕獲プローブにハイブリダイズする。 </li><li> 2.5％BSAおよび0.25＆を含む1Mのリン酸緩衝液中でオリゴヌクレオチドを​​ブリッジ1nmの4μlを、pHが7.2を適用μ、Mフルオレセイン変性検出プローブ2陽性対照センサ（信号正規に使用される）と15分間インキュベートする。 </li><li> 5秒のために、窒素気流下2-3秒、乾燥のために脱イオンH 2 Oとセンサチップを洗ってください。 </li><li> 15分間のすべての16のセンサーとインキュベートの作用電極に0.5％カゼインを含む1 Mリン酸緩衝液で0.5 U / mlの抗フルオレセイン-HRP4μlの、pHが7.2を適用します。抗フルオレセイン-HRPは、固定化されたフルオレセイン修飾された検出プローブに結合する。 </li><li> 5秒のために、窒素気流下2-3秒、乾燥のために脱イオンH 2 Oとセンサチップを洗ってください。 </li><li>センサーチップマウントにセンサチップと負荷の表面によくフィルムステッカーを適用します。全16センサーへのTMB基質のピペットを50μlとセンサチップマウントを閉じます。 </li><li>ヘリオスチップリーダーを使用しているすべての16センサー用アンペロメトリーとサイクリックボルタンメトリー測定値を得る。電流測定電流は、TMB、rの速度に比例しているセンサー表面に析出（ 図1参照）。 </li></ol>

<ol>
	<li>Wu, J., Campuzano, S., Halford, C., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+Surface+Monolayers+for+Ultrasensitive+(Zeptomole)+Amperometric+Detection+of+Nucleic+Acid+Hybridization+without+Signal+Amplification.">Ternary Surface Monolayers for Ultrasensitive (Zeptomole) Amperometric Detection of Nucleic Acid Hybridization without Signal Amplification.</a> Anal. Chem. 82, 8830-8837 (2010).</li><li>Halford, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Antimicrobial+Susceptibility+Testing+by+Sensitive+Detection+of+Precursor+Ribosomal+RNA+Using+a+Novel+Electrochemical+Biosensing+Platform.">Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor Ribosomal RNA Using a Novel Electrochemical Biosensing Platform.</a> Antimicrob. Agents Chemother. 56, (2012).</li><li>Campuzano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+monolayers+as+DNA+recognition+interfaces+for+direct+and+sensitive+electrochemical+detection+in+untreated+clinical+samples.">Ternary monolayers as DNA recognition interfaces for direct and sensitive electrochemical detection in untreated clinical samples.</a> Biosens. Bioelectron. 26, 3577-3584 (2011).</li><li>Gau, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+molecular+analysis+without+nucleic+acid+amplification.">Electrochemical molecular analysis without nucleic acid amplification.</a> Methods. 37, 73-83 (2005).</li><li>Patel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+Specific+Capture+Enhances+Sensitivity+of+Electrochemical+Detection+of+Bacterial+Pathogens.">Target Specific Capture Enhances Sensitivity of Electrochemical Detection of Bacterial Pathogens.</a> J. Clin. Microbiol. 49, 4293-4296 (2011).</li><li>Mastali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+probe+length+and+target+location+for+electrochemical+detection+of+selected+uropathogens+at+ambient+temperature.">Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature.</a> J. Clin. Microbiol. 46, 2707-2716 (2008).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrochemical+DNA+biosensors+for+rapid+molecular+identification+of+uropathogens+in+clinical+urine+specimens.">Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.</a> J. Clin. Microbiol. 44, 561-570 (2006).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+advanced+electrochemical+DNA+biosensor+for+bacterial+pathogen+detection.">Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection.</a> J. Mol. Diagn. 9, 158-168 (2007).</li><li>Pedrero, M., Campuzano, S., Pingarron, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroanalytical+sensors+and+devices+for+multiplexed+detection+of+foodborne+pathogen+microorganisms.">Electroanalytical sensors and devices for multiplexed detection of foodborne pathogen microorganisms.</a> Sensors (Basel). 9, 5503-5520 (2009).</li><li>Kuralay, F., Campuzano, S., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+disposable+nucleic+acid+biosensors+for+direct+bioelectronic+detection+in+raw+biological+samples.">Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples.</a> Talanta. 85, 1330-1337 (2011).</li><li>Ecker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ibis+T5000:+a+universal+biosensor+approach+for+microbiology.">Ibis T5000: a universal biosensor approach for microbiology.</a> Nat. Rev. Microbiol. 6, 553-558 (2008).</li><li>Casalta, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+LightCycler+SeptiFast+test+in+the+rapid+etiologic+diagnostic+of+infectious+endocarditis.">Evaluation of the LightCycler SeptiFast test in the rapid etiologic diagnostic of infectious endocarditis.</a> Eur. J. Clin. Microbiol. Infect. Dis. 28, 569-573 (2009).</li></ol>

すべての著者は説明した方法に関連する特許に関する発明である。これらの特許の一つはQvella株式会社にライセンス供与されています。 BMCとDAHはQvella株式会社の持分を持っている。 VGは、電気化学センサアレイチップのメーカーですGeneFluidicsの社長、チップマウント、そして本稿で述べたチップリーダーです。

ここで説明する電気化学センサアッセイは、核酸標的の迅速な検出を可能にする。感度および特異性は、順番に長さ及び捕捉および検出プローブのGC含有量に依存するターゲットプローブハイブリダイゼーションの自由エネルギーに部分的に依存する。我々は一般的に、周囲温度（〜20℃）5、6でのハイブリダイゼーションの手順を実行します。チップを含有する被覆チャンバ内に配?...

細菌の検出および同定のための標的分子としてのrRNAを使用すると、多くの利点を有する。細菌細胞内のrRNAの存在量が標的増幅1を必要とせずにミリリットルあたり250バクテリアの低感度限界のために用意されています。細菌のリボソームRNAは、DNAプローブとのハイブリダイゼーションにアクセス可能なユニークな種特異的な配列を含む。これにより、電気化学センサのアレイは、各センサが異なる種特異的捕捉プローブで官能化されている未知の細菌を同定するために用いることができる。ポジティブコントロールセンサーは、 &quot;ブリッジ&quot;捕捉および検出プローブが内部校正信号を作成するために合成オリゴヌクレオチド標的のために含まれるべきである。 電気化学センサーは、基本的な研究および翻訳の幅広い用途を有する。例えば、ここに記載されたアッセイを正確E.の効果を測定するために使用されているRRN上の大腸菌増殖期細菌の生理学2に興味を持って研究者に大きな関心であると事前rRNAのコピー数、。電気化学センサアッセイの感度は、信号対雑音比によって決定される。信号増幅及びノイズ低減の様々な方法が検討されている。私たちは、センサー表面の化学的性質を改善することは検出プローブおよび/またはHRP酵素の非特異的結合を減少させるための鍵であることがわかります。特に、アルカンジチオール及びメルカプトヘキサノールとの混合単分子膜は、標的ハイブリダイゼーション3に対して捕捉プローブのアクセス可能性を保持しながらより完全に電極表面を被覆することによってバックグラウンドを低減することが見出された。これらの表面化学処理は、複雑な生物学的サンプルを伴うアッセイのために特に重要である。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>6-mercapto-1-hexanol (MCH)</td>
 <td>Sigma</td>
 <td>451088</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>1,6-hexanedithiol (HDT)</td>
 <td>Sigma</td>
 <td>H-12005</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>Thiolated capture probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Fluorescein-modified detector probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Bridging Oligonucleotide</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-Fluorescein-HRP, Fab fragments</td>
 <td>Roche</td>
 <td>11 426 346 910</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Helios Chip Reader</td>
 <td>GeneFluidics</td>
 <td>GFR-2009</td>
 <td></td>
 </tr>
 <tr>
 <td>Sensor Chip Mount</td>
 <td>GeneFluidics</td>
 <td>GFR-003</td>
 <td></td>
 </tr>
 <tr>
 <td>Film well sticker</td>
 <td>GeneFluidics</td>
 <td></td>
 <td>Shipped with sensor chips</td>
 </tr>
 <tr>
 <td>Bare gold 16-sensor array chips </td>
 <td>GeneFluidics</td>
 <td>SC1000-16X-B </td>
 <td>Store in 100% N2 at room temperature</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin </td>
 <td>Sigma</td>
 <td>A7906 </td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>1M Phosphate Buffer, pH 7.2 </td>
 <td></td>
 <td></td>
 <td>0.35M NaH2PO4, 0.65M K2HPO4, adjusted to pH 7.2</td>
 </tr>
 <tr>
 <td>Blocker Casein in PBS </td>
 <td>Pierce</td>
 <td>37528 </td>
 <td>Dilute with an equal volume of 1M Phosphate Buffer, pH 7.2, store at 4 &deg;C</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents and Equipment.</td>
 </tr>
 </tbody>
</table>

bacterial detection &amp; identification using electrochemical sensors

電気化学センサが広く血糖の迅速かつ正確な測定のために使用され、検体の多種多様な検出のために適合させることができる。電気化学センサは、有用な電気信号に生体認識イベントに形質導入​​することによって動作する。シグナル伝達は、電流測定電極に酸化還元酵素の活性を結合することによって起こる。センサの特異性は、酵素の固有の特性、グルコースセンサの場合、グルコースオキシダーゼ、又は酵素及び抗体又はプローブ間のリンケージの積である。 ここでは、直接細菌を検出し、識別するための電気化学センサの測定方法を説明する。いずれの場合も、ここで説明するプローブは、DNAオリゴヌクレオチドである。この方法は、標的リボソームRNA（rRNAの）とキャプチャーと検出プローブのサンドイッチハイブリダイゼーションに基づいています。検出プローブがhに連結されている間に捕捉プローブは、センサー表面に固定されているorseradishペルオキシダーゼ（HRP）。例えば、3,3 &#39;、5,5&#39; - テトラメチルベンジジン（TMB）などの基板は、その表面に結合した捕捉対象検出器複合体を有する電極に追加されると、基板は、HRPにより酸化され、作用電極によって低減される。電極で電流の流れを作り出す、電極からHRPに基板による電子の往復でこの酸化還元サイクル結果。

私たちは、サンドイッチELISAと同様に構成されている電気化学的測定法について説明します。 図1に示すように、捕捉および検出プローブと標的リボソームRNA（rRNAの）ハイブリダイゼーションを検出プローブ上の3 &#39;フルオレセイン結合に結合する抗フルオレセイン抗体フラグメントに結合したHRPにより触媒される酸化還元反応によって開発されている。分析感度の重要な構成?...

Watch this Scientific Journal Video about Bacterial Detection & Identification Using Electrochemical Sensors at JoVE.com

Bacterial Detection &amp; Identification Using Electrochemical Sensors

我々は、迅速な菌の検出および同定のための電気化学センサの測定方法を説明する。アッセイは、リボソームRNA（rRNAの）種特異的配列のためのDNAオリゴヌクレオチド捕捉プローブで官能化されたセンサアレイを含む。捕捉プローブと西洋ワサビペルオキシダーゼ連動DNAオリゴヌクレオチドの検出プローブでターゲットのrRNAのサンドイッチハイブリダイゼーションは、測定電流測定電流を生成する。

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 ...

bacterial-detection-identification-using-electrochemical-sensors

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Bioengineering

28.1K ビュー.  Veterans Affairs Greater Los Angeles Healthcare System. 我々は、迅速な菌の検出および同定のための電気化学センサの測定方法を説明する。アッセイは、リボソームRNA（rRNAの）種特異的配列のためのDNAオリゴヌクレオチド捕捉プローブで官能化されたセンサアレイを含む。捕捉プローブと西洋ワサビペルオキシダーゼ連動DNAオリゴヌクレオチドの検出プローブでターゲットのrRNAのサンドイッチハイブリダイゼ...

ビデオ: Bacterial Detection & Identification Using Electrochemical Sensors

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

二次エレクトロスプレーイオン化質量分析法（SESI - MS）を用いて細菌揮発性物質の特性評価

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

生物工学

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. ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

光音響流量測定法を用いて循環メラノーマ細胞の検出と分離

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

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 ...

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 ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

遺伝的にコードされたセンサを用いたグルタミンフラックスイメージング

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

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 ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

安価なセンサで昆虫の検出と分類をフライング

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

マイクロトロイドの光共振器を用いたラベルフリー単一分子検出

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...