这项工作是由以色列科学基金会（批准号:1118年至1108年，并批准号1146至1112年）和明娜克罗尔纪念研究基金的支持。 ES衷心感谢罗素贝里纳米技术研究所的资金支持。

1。氧化多孔SiO 2的制备<ol><li>蚀刻Si晶片（单面抛光的&lt;100&gt;面与重掺杂的p型，0.0008Ω·cm的）在HF水溶液和无水乙醇中进行30秒的3:1（V / V）溶液在恒定电流密度为385毫安/厘米2。请注意，HF是高腐蚀性的液体，它应该格外小心。 </li><li>冲洗所得的多孔Si（PSI）膜与无水乙醇的表面数次;干燥在干燥氮气气体的薄膜。 </li><li>氧化在管式炉的新鲜蚀刻PSI的样品在800℃下1小时，在环境空气中（放置在炉中的样品在室温下，将炉子加热到800℃，在炉中离开1小时后，关掉炉，并从炉仅在室温下取出样品）。 </li></ol> 2。的PSIO 2支架Biofunctionalization <ol><li>孵育氧化PSI（防扩散安全倡议O 2）样品1小时的巯基（三甲氧基硅烷，3）95％（MPTS）溶液（108毫米的甲苯溶液）。 </li><li>冲洗硅烷处理PSIO 2样品用甲苯，甲醇和丙酮;干燥在干燥氮气气体。 </li><li>在1ml的100mM的PEO-碘代乙酰基生物素溶液孵育硅烷改性PSIO 2样品30分钟。 </li><li>冲洗生物素-治疗PSIO 2样品用0.1M磷酸盐缓冲盐水（PBS）溶液数次。 </li><li>在1ml的100微克/毫升链霉素（SA）溶液孵育生物素修饰的PSIO 2样品30分钟。 </li><li>冲洗所述SA处理的PSIO 2样品用0.1M PBS溶液数次。 </li><li>在1ml的100微克/毫升生物素化的大肠杆菌孵育所得的SA-改性PSIO 2样品30分钟大肠杆菌单克隆抗体（免疫球蛋白G，免疫球蛋白G）溶液或用100微克/毫升生物素化兔IgG（作为一种模式的单克隆抗体）。 </li></ol> 3。荧光标记和荧光显微镜<ol><li>孵育IgG的改性表面用15微克/毫升的荧光素（FITC）标记的抗兔IgG为40分钟，用15μg/ ml的荧光素（FITC）标记的抗小鼠IgG作为对照。 </li><li>冲洗改性的样品用0.1M的PBS溶液数次。 </li><li>检查在荧光显微镜下的样品。 </li></ol> 4。细菌培养<ol><li>培养大肠杆菌大肠杆菌 K12菌在一个10毫升管用5毫升的Luria BERTANI（LB）培养基中（在1升去离子水介质组合物5克氯化钠，5克酵母提取物的，和10克胰蛋白胨）中。孵育过夜细菌晃动在37°C </li><li>通过在600nm的波长的读取光密度（OD）监测细菌的浓度。在LB培养基中过夜生长后，使用分光光度计，以确定细菌浓度读取OD 600。细胞的数量是可怕CTLY成正比的OD 600测量结果（1 OD 600 = 10 8个细胞/ ml）。 </li></ol> 5。细菌检测<ol><li>将抗体修饰的PSIO 2利落PSIO 2（对照）在一个特制的有机玻璃流通池样品。固定流动池，以确保样品的反射率是在所有的测量测量是在同一地点。 </li><li>孵育E.样本大肠杆菌 K12悬浮液（10 4细胞/毫升）处理30分钟，在室温下进行。然后通过冲洗的细胞用0.85％（重量）/ 30最小V生理盐水溶液中除去细菌悬浮液。 </li><li>监测整个实验过程中的变化的反射率的数据。所有光学测量需要在周围的水性要进行。光谱应该使用CCD分光计收集并通过应用快速傅立叶变换（FFT），如前所述25,26进行分析。 </li><li>确认细菌在存在生物传感器表面上，通过观察生物传感实验后，立即直立光学显微镜下的样本。 </li></ol>

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作者宣称没有竞争的财务权益。

一种不用标记的光学免疫传感器的基础上，PSIO 2纳米结构体（法布里-珀罗薄膜）的制造，并且其潜在适用性作为生物传感器用于细菌检测被确认。 修改和故障排除一个设计免疫传感器时的主要关注是抗体的敏感性沉积和图案化期间经受不期望的构象变化到固体基板上，这可能导致在生物传感器灵敏度31,32下降。为了尽量减少这种问题，抗体...

早期准确鉴定致病菌的是食物和水安全，环境监测，并点护理诊断1极为重要。由于传统的微生物学技术是费时，费力，缺乏检测微生物的"实时"或在实验室环境之外的能力，生物传感器不断发展，以应对这些挑战2-5。 近年来，在多孔硅（PSI）已经成为一种很有前途的平台，用于传感器和生物传感器6-20的设计。在过去的十年里许多关于PSI为基础的光学传感器和生物传感器的研究发表21,22。纳米结构PSI层典型地制作由单晶Si晶片电化学阳极蚀刻。所得到的PSI纳米材料表现出许多有利的特性，例如大的表面和自由体积，孔径大小，可控制和可调谐OPTI校准性能10,16。 PSI的层，例如光致发光8,11和白光反射型干涉7,19的光学特性，强烈受到环境条件的影响。捕获在多孔层产生的薄膜，观察到的光致发光光谱的调制或者作为反射率光谱10的波长偏移的平均折射率的变化范围内的客体分子/靶分析物。 尽管在PSI光学生物传感器技术的巨大创新，也只有在PSI的平台为细菌检测6,8,20,23-29报道很少。此外，大多数证明概念的这些研究已证实"间接"细菌检测。因此，该细胞的一般现有的裂解是必需的，提取该目标蛋白质/ DNA片段，特征为研究细菌29。我们的做法是直接捕捉目标细菌细胞到PSI生物传感器。因此，单克隆抗体，其特定于针对细菌，被固定在所述多孔表面上。通过抗体-抗原相互作用的结合细菌细胞的，固定在生物传感器的表面上引起的变化的反射光谱24-26的振幅（强度）。 在这项工作中，我们报告的光学PSI为基础的生物传感器的构造，并证明了其作为无标记生物传感平台， 大肠埃希氏菌的检测（ 大肠杆菌 ）K12菌（作为模型微生物），监控的应用光信号是从PSI纳米结构反映由于法布里-珀罗薄膜干涉（ 图1A）的光。改变光的振幅/强度是相关的，以在目标细菌细胞的特异性固定到生物传感器表面上，从而允许快速检测和细菌的定量。

<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>基于纳米结构的多孔Si一种不用标记的光学生物传感器被设计为大肠杆菌 K12细菌的快速捕获和检测，作为一个模型的微生物。在生物传感器依赖于直接结合的靶细菌细胞在其表面上，同时研究了样品的无预处理（ 例如，通过细胞裂解）是必需的。的中孔硅薄膜被用作生物传感器的光学换能器元件。在白光照明下，多孔层显示在它的反射率光谱很好分辨的法布里 - 珀罗干涉条纹图样。应用快速傅立叶变换（FFT），以在一个单一的峰值反射率数据的结果。变化的FFT峰的强度进行监测。因此，目标捕捉细菌到生物传感器表面，通过抗体 - 抗原相互作用，诱导的FFT峰的强度可测量的变化，允许一个"实时"观察细菌附着。 NT"&gt;介孔硅薄膜，通过电化学阳极氧化工艺制造，缀合的单克隆抗体，特异于靶细菌的固定化，免疫活性和抗体的特异性是由荧光标记的实验证实，一旦生物传感器暴露于靶细菌，细胞被直接捕获到的抗体修饰的多孔硅表面。这些特定的捕获事件导致在生物传感器的薄膜光学干涉光谱强度的变化。我们表明，这些生物传感器可以检测到相对低的细菌浓度（检测限制在不到一个小时10 4个细胞/ ml）。

氧化PSI（PSIO 2）膜作为在协议文本部分中所述制备。 图1B示出的热氧化后得到的PSI膜的高分辨率扫描电子显微照片。该PSIO 2层的特征在于良好定义的圆柱形孔，其直径在30-80纳米的范围内。 的单克隆抗体（IgG）分子是通过使用一个完善的硅烷化技术，加上生物素-SA系统接枝到PSIO 2的表面。详细的合成方案在图2中所概述。首先?...

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

一个无标记光学生物传感器快速检测细菌的介绍。该生物传感器是基于纳米结构的多孔硅，被设计成直接捕获目标细菌的细胞在其表面上。我们使用的单克隆抗体，固定在所述多孔性换能器，作为捕获探针。我们的研究表明这些生物传感器用于与事先没有样品处理（如细胞裂​​解）分钟内低细菌浓度的检测的适用性。

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

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 次观看.  Technion - Israel Institute of Technology. 一个无标记光学生物传感器快速检测细菌的介绍。该生物传感器是基于纳米结构的多孔硅，被设计成直接捕获目标细菌的细胞在其表面上。我们使用的单克隆抗体，固定在所述多孔性换能器，作为捕获探针。我们的研究表明这些生物传感器用于与事先没有样品处理（如细胞裂​​解）分钟内低细菌浓度的检测的适用性。 

视频: 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.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

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. ...

科研

生物工程

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.

阿的Cre - LOX P为检测细胞融合重组方法在体内</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.

将生物探针使用硅烷偶联剂的硅光学生物传感器

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.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

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.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

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.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

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.

单个RNA分子的光镊纳米操作

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.

无标记单分子检测使用微型环光学谐振器

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.

使用多色荧光检测液滴微流控光纤

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.