この作品は、イスラエル科学財団（助成番号1118から1108と認可番号1146から1112年）と、みんなのクロール記念研究基金によってサポートされていました。 ESは感謝ラッセルBerrieのナノテクノロジー研究所の財政支援を認めるものです。

1。酸化された多孔性のSiO 2の調製<ol><li>定電流で30秒間水性HFおよび無水エタノールの3:1（v / v）の溶液中でエッチングSiウエハ（片面が&lt;100&gt;面に研磨し、高濃度にドープされたp型、0.0008Ω·cm）で385ミリアンペア/ cm 2の密度。 HFは腐食性の高い液体であり、それは、細心の注意を払って処理する必要がありますのでご注意ください。 </li><li>無水エタノールで得られた多孔質Siの（PSI）膜の表面を数回すすぎ、乾燥窒素ガス下でフィルムを乾燥させる。 </li><li> （オフにし、1時間、炉内に残して、800℃まで炉を加熱し、室温で炉内にサンプルを置き、周囲の空気中で1時間、800℃で管状炉中の新たエッチングのPSIサンプルを酸化炉のみ室温で炉からサンプルを削除します）。 </li></ol> 2。 PSIO 2足場の生体機能化<ol><li>酸化したPSi（PSIをインキュベートメル中で1時間（トリ-3）95％のO 2）のサンプル（MPTS）溶液（トルエン中108 mm）である。 </li><li>トルエン、メタノール、アセトンでシラン処理PSIO 2サンプルをすすぎ、乾燥窒素ガス下で乾燥させます。 </li><li> 100 mMのPEO-ヨードアセチルビオチン溶液を1ミリリットル中に30分間シラン変性PSIO 2サンプルをインキュベートする。 </li><li> 0.1Mのリン酸緩衝生理食塩水（PBS）溶液で数回、ビオチン処理PSIO 2サンプルを洗浄します。 </li><li> 100μg/ mlのストレプトアビジン（SA）の溶液1ミリリットル中に30分間ビオチン修飾PSIO 2サンプルをインキュベートする。 </li><li> 0.1MのPBS溶液で数回、SA処理PSIO 2サンプルを洗浄します。 </li><li> 100μg/ mlのビオチン化Eの1ミリリットル中に30分間その結果、SA-修正PSIO 2サンプルをインキュベート大腸菌モノクローナル抗体（免疫グロブリンG、IgG）の溶液または100μg/ mlのビオチン化ウサギIgGで（モノクローナル抗体のモデルとして）。 </li></ol> 3。蛍光標識し、蛍光顕微鏡<ol><li> 15μg/ mlのフルオレセイン（FITC）でIgGを修飾された表面をインキュベート40分間抗ウサギIgGをタグ付け15μg/ mlのフルオレセイン（FITC）でコントロールとして抗マウスIgGタグ付き。 </li><li> 0.1MのPBS溶液で数回変更されたサンプルを洗浄します。 </li><li>蛍光顕微鏡下でサンプルを調べる。 </li></ol> 4。細菌培養物<ol><li> Eを養う大腸菌 K12（1Lの脱イオン水で培地組成：NaClを5gの酵母エキス5g、及びトリプトン10g）をルリアベルターニ（LB）培地5mlで10mlチューブ中の細菌。細菌は一晩37℃で振とう培養する</li><li>波長600nmでの光学密度（OD）を読み取ることにより、細菌濃度を監視します。 LB培地で一晩増殖させた後、細菌濃度を決定するために分光光度計を用いてOD 600を読む。細胞の数は悲惨ですOD 600の測定値（1 OD 600 = 10 8個/ ml）にCTLY比例。 </li></ol> 5。細菌検出<ol><li>カスタムメイドのプレキシグラスフローセル中のIgGで修飾されたPSIO 2できちんとPSIO 2（対照として）サンプルを置きます。サンプルの反射率は、全ての測定中に同じ場所で測定されることを確実にするために、フローセルを修正します。 </li><li> Eでサンプルをインキュベート大腸菌 K12懸濁液（10 4細胞/ ml）を、室温で30分間インキュベートした。その後、30分間のW / V食塩水0.85％でセルをフラッシュすることによって、細菌懸濁物を除去します。 </li><li>実験を通して反射率データの変化を監視する。全ての光学測定は、周囲の水性中で実施される必要がある。先に説明したように25,26スペクトルは、CCDスペクトロメーターを用いて収集し、高速フーリエ変換（FFT）を適用することによって分析されるべきである。 </li><li>上の細菌の存在を確認バイオセンサ表面は、すぐにバイオセンシング実験後の直立光顕微鏡下での試料の観察による。 </li></ol>

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著者らは、競合する経済的利益を宣言していません。

PSIO 2ナノ構造（ファブリペロー薄膜）に基づくラベルフリー光学免疫センサーは、製造されており、細菌検出用バイオセンサーとしての適用可能性が確認された。 修正とトラブルシューティング免疫センサの設計の主要な関心事の一つは、バイオセンサの感度31,32の低下につながる可能性固体基板上に堆積及びパターニング中の望まし...

病原菌の早期かつ正確な識別は、食品や水の安全性、環境モニタリング、およびポイントオブケア診断の1のために極めて重要である。伝統的な微生物学技術は時間がかかり、面倒であり、「リアルタイム」で微生物を検出する能力を欠いているか、実験室環境の外ように、バイオセンサーは、これらの課題2-5を満たすように進化している。 近年では、多孔質Si（PSI）は、センサーやバイオセンサー6月20日の設計のための有望なプラットフォームとして浮上している。過去十年間PSI-ベースの光学センサーやバイオセンサーに関する多くの研究は、21,22を発表した。ナノ構造のPSi層は、典型的には単結晶Siウェハからの電気化学陽極エッチングにより作製される。ナノ材料は、このような大規模な表面と、自由体積など多くの有利な特性を示し、その結果、PSI制御され、調整可能な最適可能な孔の大きさCALの特性10,16。このような光ルミネ8,11と白の光の反射率に基づく干渉7,19などのPSI層の光学特性は、強く、環境条件の影響を受けている。フォトルミネッセンススペクトルの変調として又は反射率スペクトル10の波長シフトとして観察されたフィルムの平均屈折率の変化、多孔質層における結果内でゲスト分子/標的分析物の捕捉。 PSiは、光バイオセンサー技術の広大な革新が、細菌検出6,8,20,23-29ためのPSI-ベースのプラットフォームでのみ報告は少ない。さらに、これらの概念実証研究のほとんどは、「間接的」細菌検出を実証した。したがって、細胞の溶解は、一般的に従来検討細菌に特徴的な29、標的タンパク質/ DNA断片を抽出するために必要とされる。我々のアプローチを直接標的細菌を捕捉することであるのPSIバイオセンサーへの細胞。したがって、細菌を標的とする特異的であるモノクローナル抗体は、多孔質表面上に固定化される。抗体-抗原相互作用を介して、細菌細胞の結合は、バイオセンサーの表面に反射率スペクトル24-26の振幅（強度）の変化を誘導する。 本研究では、光PSI-ベースのバイオセンサーの構築に報告し、 大腸菌の検出（モデル微生物として使用される）（ 大腸菌 ）K12菌用のラベルフリーバイオセンシングプラットフォームとしての用途を示しています。監視対象光信号は、光によるファブリペロー薄膜干渉（ 図1A）へのPSiナノ構造からの反射である。光振幅/強度の変化は、細菌の迅速な検出および定量を可能にする、バイオセンサー表面上に標的細菌細胞の特異的な固定化に相関している。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Siltronix Corp.</td>
			<td></td>
			<td>Highly-B-doped, p-type, 0.0008 &#937;-cm resistivity, &#60;100&#62; oriented</td>
		</tr>
		<tr>
			<td>Aqueous HF (48%)</td>
			<td>Merck</td>
			<td>101513</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>818760</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>Saline 0.85% w/v</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>95% (3-Mercaptopropyl)trimethoxysilane (MPTS)</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-iodoacetyl biotin</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>B2059</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (SA)</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-000-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein (DTAF)-streptavidin</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-010-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>011-060-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>111-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>115-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated E. coli antibody</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli (K-12)</td>
			<td></td>
			<td></td>
			<td>was generously supplied by Prof. Sima Yaron, Technion</td>
		</tr>
	</tbody>
</table>

optical detection of e. coli bacteria by mesoporous silicon biosensors

<html>ナノ多孔質Siに基づくラベルフリー光学バイオセンサーは、モデル微生物として、 大腸菌 K12細菌の迅速な捕捉および検出のために設計されています。研究された試料の前処理なし（細胞溶解などにより ） が必要とされないながら、バイオセンサーは、その表面に標的細菌細胞の直接結合に依存しています。メソポーラスSi薄膜は、バイオセンサの光変換素子として使用される。白色光照明の下では、多孔質層は、その反射率スペクトルでよく解像ファブリ·ペロー干渉縞を表示する。高速フーリエ変換は、単一のピーク反射率データの結果に変換（FFT）を適用する。 FFTピークの強度の変化が監視される。したがって、標的細菌がバイオセンサー表面上に捕獲抗体 - 抗原相互作用を介して、細菌付着の「リアルタイム」の観察を可能にする、FFTのピークの強度における測定可能な変化を誘導する。 ntの &quot;&gt;電気化学的陽極酸化法により製造されたメソポーラスSi膜は、標的細菌に特異的なモノクローナル抗体とコンジュゲートされている。抗体の固定化、免疫活性および特異性を、蛍光標​​識実験により確認されている。バイオセンサーが曝露されると標的細菌は、細胞を直接抗体修飾多孔Si表面上に捕捉され、これらの特異的捕捉のイベントは、バイオセンサーの薄膜光干渉スペクトルの強度変化をもたらす。我々は、これらのバイオセンサ（検出比較的低い細菌濃度を検出することができることを実証時間未満で10 4細胞/ ml）の上限。

プロトコルのテキストセクションに記載したように酸化されたPSi（PSIO 2）膜が調製される。 図1Bは、熱酸化後に得られたPSi膜の高分解能走査電子顕微鏡写真を示す。 PSIO 2層は30〜500nmの範囲の直径を有する明確に定義された円筒状の細孔によって特徴付けられる。 モノクローナル抗体（IgG）の分子は、ビオチン-SAシステムと結合された十分に...

Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

迅速な細菌検出用ラベルフリー光学バイオセンサーを導入する。バイオセンサーは、直接的にその表面上に標的細菌の細胞を捕捉するように設計されているナノ多孔質Siに基づいている。私たちは、捕捉プローブとして、多孔質のトランスデューサに固定化モノクローナル抗体を使用しています。我々の研究は、（例えば、細胞溶解など）なし優先検体処理に数分以内に、低濃度の細菌の検出のためのこれらのバイオセンサーの適用可能性を実証する。

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 ...

optical-detection-of-e-coli-bacteria-by-mesoporous-silicon-biosensors

Research

JoVE Journal

Bioengineering

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

16.8K ビュー.  Technion - Israel Institute of Technology. 迅速な細菌検出用ラベルフリー光学バイオセンサーを導入する。バイオセンサーは、直接的にその表面上に標的細菌の細胞を捕捉するように設計されているナノ多孔質Siに基づいている。私たちは、捕捉プローブとして、多孔質のトランスデューサに固定化モノクローナル抗体を使用しています。我々の研究は、（例えば、細胞溶解など）なし優先検体処...

ビデオ: Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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

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

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

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

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

生物工学

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 ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

シランカップリング剤を用いた石英系光バイオセンサーへの生物学的プローブの取り付け

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

マルチスペクトルイメージングフローサイトメトリーによるナノ粒子と細菌の細胞内在化を分析する

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

抗生物質耐性ブドウ球菌の細菌の検出のためのバイオセンサー

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

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 ...

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 ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.