This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.

1。実験のセットアップとサンプルの調製<ol><li>以前に6説明したように、リソグラフィ、エッチング、および融解手順を使用して、マイクロトロイドを製作。一般的に80〜100ミクロンの主要な直径を有するシリコンウェハ（チップ）の上にマイクロトロイドを製作し、2μmの短径。 </li><li>その繊維スプールからおよそシングルモード光ファイバ（125μmのクラッド、4.3ミクロンのモードフィールド径）のメーターをおくつろぎください。 </li><li>光ファイバの巻き出し部分の途中に、ワイヤーストリッパーを使用して光ファイバの周りにポリマーコーティングの小部分（2.5センチ）ストリップ。注：これは、マイクロトロイドにエバネッセント光を結合するために使用される光ファイバの一部です。 </li><li>イソプロパノールアルコールとフリーワイプリントでストリップ繊維をきれいにしてください。 </li><li>磁気クランプ製のファイバホルダを使用して所定の位置に光ファイバのこの部分を持ってください。 </li><li> MELTINによって〜500 nmの薄い取り除か繊維グラム水素トーチ60ミクロン/分の速度で反対方向に動く2ステッピングモータを使用して引っ張ります。 〜10ミリメートル背が高くあるべきである炎の上部、内部に光ファイバを配置します。ファイバを通して送信は（オフに繊維点滅から横方向に散乱した光を見て）、またはオシロスコープに接続されているフォトダイオードに光ファイバを接続することにより、視覚的に監視することのできる、変動停止したときに、繊維を引っ張って停止。 </li><li>光ファイバの一端を切断し、裸ファイバアダプタに挿入します。受光の入力に繊維のこの端を置きます。 </li><li>光ファイバカプラを使用してレーザーのファイバーカップル繊維スプールのもう一方の端を。 </li><li>エポキシ又は両面テープのいずれかを使用して試料ホルダーの上にマイクロトロイドチップ（ステンレス鋼37.8ミリメートル×6.4ミリメートルX 3.2ミリメートル）を配置します。 </li><li> 3軸ナノポジショニング（Nで構成位置決めステージの上に試料ホルダーをマウント3軸マイクロメータの上anocube）ステージ（機器リストを参照してください）​​。振動を最小限に抑えるために、空気圧で分離された光学テーブル上のすべての実験を行います。 </li><li>粗位置3軸マイクロメータを使用して、サンプルチップ。 </li><li>ナノポジショナーを使用して光ファイバにマイクロトロイド含有チップ並列の位置を合わせます。注：入力光（〜633 nm）での1波長の距離内にマイクロトロイドの位置を合わせます。このプロセスの可視化のための2つの撮像列使用上、チップの側に位置する（対物レンズとカメラとチューブを、機器リストを参照してください）​​。 </li><li>偏光を調整するつまみで（機器のリストを参照）、インライン偏光制御器を用いた光ファイバを介して照射されるレーザ光の偏光を最適化します。注意：光ファイバの伝送で測定されたディップが狭く表示されたときに最適な偏光が達成されます。 （詳細については、ステップ2.2を参照）、オシロスコープ上でこのディップを守ってください。 </li><li>構成しますスペーサーとしてガラス顕微鏡スライドを用いて、試料ステージの上にカバーガラスをepoxyingにより試料室。注：全体のセットアップを覆うプレキシグラスエンクロージャは、空気の流れを最小化するために有用であり得ます。小さな開口部は、チューブを使用してポンピングするためのソリューションを可能にするようにしておく必要があります。 </li><li>熱RT水浴（〜500 mL）中≥1時間粒子懸濁液または単一分子の水性溶液を平衡化。注：サンプルは、メーカー、 例えば 、PBSまたはHEPESから指定された関連したバッファを使用してマイクロ遠心チューブに所望の濃度に希釈します。いかなる結合事象が検出されない場合、緩衝液の塩濃度を増加させます。 </li><li> 〜2秒のためのソリューション（1ミリリットル）を簡単に含む渦粒子。 </li><li> 1mlシリンジポンプを用いて1ml /分で試料チャンバに溶液を含む粒子を注入します。 </li><li>試料室を充填した後、シリンジポンプをオフにします。 </li><li>最小限に抑えるために、データを記録する前に30秒待ってください測定に影響を与えることができ、流体の流れに起因する機械的振動の影響。 </li></ol> 2.周波数のロック<ol><li>カップリングが原因で流体の注入に邪魔されるため、ナノポジショナーと試料ホルダーを移動させることにより、光ファイバに再結合しトロイド。 </li><li>様々な波長を介してコンピュータ制御入力レーザを走査することにより、マイクロトロイドの共振波長の位置を確認します。レーザの波長を調整するレーザコントローラ内の圧電素子に三角波電圧信号を送信することによって、この手順を実行します。この波長での水の光の低い吸収があるため、可視光（2.5 nmの±635 nm）を用いた実験を行います。 </li><li>オートバランス受光に光ファイバの出力を接続することにより、光ファイバを介して光透過率を測定します。 BNCケーブルを使用して、オシロスコープに受光の出力を接続します。 O観察nはマイクロトロイドの共振波長において、光ファイバを介して伝送が低下オシロスコープ。 </li><li>ケーブルを介して（機器のリストを参照）、周波数ロックフィードバック制御装置の主入力に受光の出力を接続します。 </li><li> 2 kHzから19 FMの波長振動の振幅のディザリング周波数を持つトップのピークロックを使用して、オートロックモードでフィードバック制御をロックする周波数を実行します。経験的に、チーグラー・ニコルスのチューニング規則14を使用して、ソフトウェアのウィンドウで、比例・積分・微分設定値を設定します。注：これらの値は、すべての実験の開始時に一度設定するだけで済みます。 </li><li>マイクロトロイドの共振波長に対するレーザの波長を自動ロック。試料室を充填した後、この手順を実行します。注：波長シフトが大きすぎる場合には、フィードバック制御がロックを失うことになると自動的に共振波長の位置を見つけるために走査モードに切り替えます。このOC波長に対するCURSは約1線幅（ここでは調査したすべてのシステムのために少なくとも600 FM）よりも大きくシフトします。 </li><li> 24ビットのデータ・アクイジション・カードを使用して、20 kHzでフィードバック制御器の出力を記録します。データ収集ソフトを経由してテキストフ​​ァイルとしてデータをエクスポートします。 </li></ol> 3.データ処理と解析<ol><li>フーリエ変換は、MATLABのデータを変換します。 </li><li>ローは、2 kHzで課されるディザ周波数を除去するために、1 kHzでカットオフを持つ「ブリックウォール」フィルターを使用してデータを渡す（補足コードスクリーンショット1ファイルを参照してください）​​。 </li><li>計算ノッチ16ヘルツのウィンドウサイズを使用してデータをフィルタリングします。注意：これは、この場合には、ノイズの既知のソースを除去するために行われ、60 Hzの電子線ノイズとその高調波、（レーザドライバからの）だけでなく、100ヘルツとその高調波（補足コードスクリーンショット1ファイルを参照してください）​​。 </li><li>逆フーリエ変換は、時間領域に戻ってデータを変換します。 </li><li>メディアンフィルタウィンドウを使用してデータ1,001サンプルの大きさ（補足コードはスクリーンショット2ファイルを参照してください）​​。 </li><li> Kerssemakers ら 15のステップ発見アルゴリズムを用いて、共振波長のステップ変化を探します。 </li><li>各結合工程の振幅のヒストグラムを生成します。 </li><li>式を使用して粒子サイズを計算します。 （1）（説明を参照）。 </li></ol>

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The authors declare that they have no competing financial interests.

粒子が結合するように、トロイドは増加の共鳴波長（λ）。粒子がアンバインドした場合、共振波長は、それに応じて（ステップダウンイベント）を減少させます。粒子径（d）は、各波長ステップの振幅のヒストグラムを介して決定することができます。各波長ステップの高さは、結合した粒子の大きさの変化に起因し、粒子が結合マイクロトロイドの位置により変化。粒子が?...

単一分子検出実験は、疾患の早期発見のために、及び分子 3の基本特性を調べるために、バイオセンサーに使用される分析物の量を減少させるために有用です。このような実験は通常、ラベルを使用して実行されますが、ラベルは、特定のタンパク質を得ることが常に可能ではない、コストを増加させ、オンサイト実験やポイント・オブ・イベントが検討されて乱すことができ、かつ不便であることができ、特にリアルタイムのためのケア診断。 無標識バイオセンシングのための現在のゴールドスタンダードは、表面プラズモン共鳴4であり、ただし、商業表面プラズモン共鳴システムは、典型的には、nMの程度の検出の典型的な下限を有します。最近では、光共振器は、ラベルフリー単一分子の生物学的検出のための5有望な技術として浮上しています。光共振器は、光6,7の長期（NS）閉じ込めに基づいて動作します。光がエバネッセントです一般的に光ファイバを介してこれらのデバイスに結合されました。光ファイバを通過する光の波長が共振器の共振波長と一致すると、共振器に効率的にカップルを点灯。この結合された光は、全内部共振器の周付近にエバネッセント場を発生させる共振器の空洞内に反映されます。粒子は、共振器、 粒子 8の体積に比例して共振器の変化の共振波長にエバネセント場とバインドを入力します。 検出能力の点で、微小球共振器は、以前の（100 nm）の9,10ウイルス粒子の単一のインフルエンザを検出するために使用されてきました。最近、plasmonically増強微小球光共振器は、 分子 11と8マーのオリゴヌクレオチド12を 、単一のウシ血清アルブミンを検出するために使用されている、しかし、このアプローチは、デ0.3ミクロン2に粒子捕捉領域を制限します副。大きなキャプチャエリアのバイオセンサーは、粒子検出の可能性を最大化するための理想的です。 （&gt; 100μmの2）大と現在のソリューションベースのラベルフリーバイオセンシング技術領域をキャプチャポリスチレン粒子≥25 nmの検出に限られていました。 我々は、溶液中の単一分子の時間分解検出が可能な周波数ロック光ウィスパリングエバネッセント共振器（フラワー）13（ 図1）として知られている光共振器技術に基づくラベルフリーバイオセンシングシステムを開発しました。花がダウンして、単一のタンパク質分子に小さな粒子を検出するために、周波数ロックフィードバック制御、バランス検出、計算フィルタリングと組み合わせたマイクロトロイドの光共振器の長い光子寿命を使用しています。周波数ロックの使用は、掃引またはレーザ波長を走査する必要なしに、粒子を結合するように、システムは、常に、マイクロトロイドのシフト共鳴を追跡することができ大規模な範囲です。 FLOWERの原理は、プラズモン増強を含む​​他の技術の検出能力を向上させるために使用することができます。以下では、FLOWERを実行するための手順が記載されています。

<table border="0" cellpadding="0" cellspacing="0" width="396">
	<tbody>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Tunable diode laser</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Laser controller</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300-LN</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Frequency locking feedback controller</td>
			<td class="xl63" style="width:99pt" width="132">Toptica Photonics</td>
			<td class="xl63" style="width:99pt" width="132">Digilock 110</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Auto-balanced photoreceiver</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">Model 2007</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">In-line polarization controller</td>
			<td class="xl63" style="width:99pt" width="132">General Photonics</td>
			<td class="xl63" style="width:99pt" width="132">PLC-003-S-90</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">24-bit data acquisition card</td>
			<td class="xl63" style="width:99pt" width="132">National Instruments</td>
			<td class="xl63" style="width:99pt" width="132">NI-PCI-4461</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">Recombinant human interleukin-2</td>
			<td class="xl63" style="width:99pt" width="132">Pierce Biotechnology</td>
			<td class="xl63" style="width:99pt" width="132">R201520</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">20 nm polystyrene beads</td>
			<td class="xl63" style="width:99pt" width="132">Thermo Scientific</td>
			<td class="xl63" style="width:99pt" width="132">3020A</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">NanoCube XYZ Piezo Stage</td>
			<td class="xl63" style="width:99pt" width="132">Physik Instrumente</td>
			<td class="xl63" style="width:99pt" width="132">P-611.3</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Optical table</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">VH3660W-OPT</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Objective lens for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl64">1-60228</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Imaging column (adaptor tube)</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl65">1-60228</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">High-Res CCD camera for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Edmund Industrial Optics</td>
			<td class="xl64">NT39244</td>
		</tr>
	</tbody>
</table>

label-free single molecule detection using microtoroid optical resonators

ダウン単一分子の限界に分子の低濃度を検出することは、そのような疾患の早期発見、および分子の挙動に関する基礎的研究などの分野に影響を与えています。単一分子検出技術は、一般に、蛍光タグまたは量子ドットなどのラベルを使用し、しかし、ラベルは常に利用可能ではない、コストと複雑さが増大し、研究されているイベントを乱すことができます。光共振器は、ラベルを使用せずに単一分子を検出するための有望な手段として浮上しています。現在、溶液中の非plasmonically増強裸光共振器システムによって検出された最小粒子が25nmのポリスチレン球1です。私たちは、この制限を超えると水溶液2のラベルフリーの単一分子検出を達成することができ、周波数ロック光ウィスパリングエバネッセント共振器（フラワー）と呼ばれる技術を開発しました。信号強度は粒子体積に比例するように、私たちの仕事は、&gt; 100倍improvemeを表しNT現在の技術水準を超える信号対雑音比（SNR）です。ここFLOWERの背後にある手順は、フィールドでの使用を高めるために提示されています。

粒子結合事象は明らかに時間をかけてマイクロトロイドの共振波長の階段状の変化（ 図2A）と見られています。これらのステップの高さは。 図2Bのグラフとして示さ2-4はそれぞれ、代表エキソソームの結合からのトレース（nanovesicles）、5 nmのシリカビーズ、および単一のヒトインターロイキン2分子を示しています。階段状のイベントは、粒...

Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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 ...

label-free-single-molecule-detection-using-microtoroid-optical

Research

JoVE Journal

Bioengineering

9.1K ビュー.  California Institute of Technology. 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. 

ビデオ: Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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 ...

生物工学

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

細胞融合の検出のためのCre - LOX Pの組換えのアプローチインビボでの</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

ラマン分光法を用いた細胞と細胞外マトリックスの非接触、ラベルフリーの監視

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

を光学的に検出<em&gt; E.大腸菌の</emメソポーラスケイ素バイオセンサーによる&gt;バクテリア

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Gタンパク質共役受容体によるカリウムチャネルの調節を検出するために、ラベルフリー光学バイオセンサの使用

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

光ピンセットによる単一のRNA分子のナノマニピュレーション

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

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 ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

単一細胞分泌物の時空間イメージング用ラベルフリー技術

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

平面支持された二分子層​​上の単一分子蛍光顕微鏡

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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 ...