作者感谢养养眼博士在弗雷德Sigworth博士在耶鲁大学的HEK293细胞稳定表达大鼠斯莱克-B蛋白质的慷慨捐赠实验室。此外，作者感谢Sigworth博士在视频介绍中显示松弛的低温电子显微镜同源模型。这项研究是由美国国立卫生研究院资助DH067517和NS073943到LKK支持

1。细胞培养（从弗莱明和卡兹玛瑞克18改编） <ol><li>培养的细胞在适当的介质为大于2且小于10的通道在该试验中使用之前。未转染的HEK-293细胞和HEK-293细胞的稳定表达大鼠松弛-B蛋白质是生长在一个半Dulbecco改良的Eagle培养基中，另一半Leibovitz的L-15培养基补充有10％热灭活的胎牛血清和抗生素。 500微克/毫升的遗传霉素（G418）加入到HEK-293细胞的稳定表达松驰-B来选择的时差信道的表达。传代细胞在70-90％汇合时通过离解离的菜肴用0.25％胰蛋白酶-EDTA溶液。 </li></ol> 2。在TiO 2 384孔的光学生物传感器板与纤维连接蛋白和卵清蛋白的细胞外基质（ECM）涂层<ol><li>纤连蛋白的ECM混合物和卵清蛋白封闭液的制备<ol><li>准备fibronect的工作液在磷酸盐缓冲盐水（PBS）与20.0毫升的PBS 5微克/纤连蛋白毫升的最终浓度。从1.0毫克/毫升的浓度股票，加入100微升至20.0毫升的PBS为200倍稀释。纤连蛋白的工作液可以在制备后进行等分并储存在-20℃。 </li><li>制备在无菌水热失活的卵清蛋白的5％的原液。在无菌组织培养级水稀释卵清蛋白为5％的溶液，并在60℃下热灭活30分钟冷却该溶液至室温，并无菌过滤的溶液。分装在4℃的5％卵清蛋白溶液与存储</li><li>通过稀释5％的储备溶液至PBS制备的卵清蛋白的2％工作液。例如，添加12.0毫升的5％卵清蛋白原液到18.0毫升的PBS为30.0毫升的最终体积。 </li></ol></li><li> 384孔光学生物传感器板涂层<ol><li>与20.0微升/孔的水合TiO 2的板的无菌组织培养级水中15分钟。下面的步骤应该具有16通道移液器进行。 </li><li>与50.0微升PBS，每孔洗板1X。 </li><li>添加20.0微升的纤连蛋白工作液到每个孔中并孵育2小时，在室温下创建的ECM。 </li><li>与50.0微升PBS，每孔洗板1X。 </li><li>添加40.0微升卵清蛋白工作液到每个孔中并孵育过夜，在4℃阻断板。 </li><li>与50.0微升PBS，每孔洗板3倍。 ECM包被的板可以被存储在4℃下长达48小时。 </li></ol></li></ol> 3。细胞接种到384孔的光学生物传感器板<ol><li>从细胞中除去生长培养基，并添加胰蛋白酶到从板移去细胞。收集细胞并离心以1,000×g离心1分钟。取出胰蛋白酶媒体和悬浮细胞在生长培养基。 </li><li>使用血细胞计数器或自动细胞计数，测定细胞的浓度。 </li><li>以减少蒸发的温育过程中的影响，50.0微升每孔的生长培养基将被使用。细胞应稀释至达到每孔细胞中的50.0微升生长培养基的体积所需的号码。对于这些实验，1,000个细胞/孔接种到生物传感器。 </li><li>使接种的细胞在空气/ 5％CO 2下过夜孵育在生长培养基中，在37℃。 </li></ol> 4。生物传感器和复合板的RWG光学生物传感器测定的制备<ol><li>从培养箱中取出盘子并从细胞中除去生长培养基。洗涤细胞1倍，用Hanks平衡盐溶液（HBSS）中。添加25.0微升HBSS中的每个孔中。 </li><li>将生物传感器板上的BIND扫描器和使细胞平衡至室温1-2小时。 </li><li>准备一个单独的复合板从利益的配体将是广告在试验过程中副执行干事到生物传感器板。对于这些测定，25.0微升含有溶液的化合物加入到每个孔中的生物传感器板为50.0微升的总体积。因此，在溶液中的化合物的2倍的所需最终浓度制备。 </li></ol> 5。执行RWG光学生物传感器测定<ol><li>采取的基线测量使用的3.75微米/像素的最大分辨率为中央1.5毫米每个使用BIND扫描软件很好的扫描仪系统，用于井A1-P12（在SBS 384孔板的一半）。这个基线扫描大约需要30分钟。 </li><li>添加25.0微升的化合物溶液，每孔为孔A1-P12。 </li><li>利用井A13-P24基线测量。 </li><li>添加25.0微升的化合物溶液，每孔为孔A13-P24。 </li><li>对井A1-P12后加入化合物计量，并重复井A13-P24。 </li></ol> 6。一化验数据的na​​lysis与BIND查看<ol><li>打开Bind分析数据，可在Windows的基线测量，并单击“网格编辑器”按钮，使小区选择。该软件将计算出由用户定义的兴趣量度。 </li><li>打开“插件”，其中包括“手机搜索”和“底线型”功能。在“小区搜索”设置参数，使得细胞是来自生物传感器的背景区分开来。 </li><li>导出其中包含“手机搜索”的数据为基准测量单元映射文件。 </li><li>重复步骤6.1-6.2对复合后的另外的数据。 </li><li>打开“底线型”插件，并从基线测量导入细胞的地图。该软件将计算出的背景和复合后的数据之间的转变PWV。 </li><li>选择“Δ细胞平均”的意思转变为PWV电池的输出。这些数据可以被导出到Microsoft EXCEL作进一步分析。 </li></ol>

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史蒂芬M. Shamah为X-BODY Biosciences公司的雇员。

在Z素（Z&#39;）的因素是量化的高通量筛选测定法20的质量的普通统计方法，并且因为G蛋白偶联受体激动剂在此测定法的筛选中发生一个SBS兼容384孔分析，提供了极好的衡量这个实验21的鲁棒性和有效性。 1 A-Z&#39;值表示理论上的理想实验与数据点与不存在的标准偏差无限多。 0.5-1之间AZ&#39;值被认为是一个很好的检验为高通量筛选的目的，用低于0.5被认为是轻微翔实的分析。对于这...

在他们的生理有关的上下文检查活细胞关键的是要了解目标细胞的生物学功能。然而，实验研究渠道和监管细胞质蛋白，如免疫沉淀测定法之间的相互作用，通常提供的信息很少在活细胞相互作用的时间过程。大多数当前的基于细胞的测定法测定特异性细胞事件，例如感兴趣的荧光标记蛋白的易位。这些测定中所需要的感兴趣的蛋白质的修饰，这可以潜在地改变蛋白质的行为和减少靶的生理相关性。非侵入性的基于细胞的测定法，它不需要操纵系统，提供细胞活性的连续测定，并允许细胞的研究，在其最生理学相关的状态4。 光学生物传感器的设计，生产一个可测量的变化在被耦合到所述传感器表面的光的特性。光学生物传感器，利用渐逝波，其中包括表面等离子体共振（SPR）和谐振波长光栅（RWG）技术，已被主要用于检测分子结合到其固定在传感器表面上的生物受体的亲和力和动力学。最近，市售的RWG生物传感器已被开发，以使细胞附着在传感器表面上的高空间分辨率（高达3.75微米/像素）5的底部部分内的质量的相对变化的检测。这些生物传感器可以检测到附近的活细胞所导致的刺激作用，如细 ​​胞粘附和铺展性，毒性，增殖和由多种细胞表面受体，包括G蛋白偶联受体（GPCRs）的诱发信号转导通路的质膜变化6质量。 RWG生物传感器检测的相对瓒格中折射的作为内传感器7为150nm的质量的相对变化的函数的索引。当细胞接种到生物传感器，这种变化在折射指数反映的质量变化从靠近在蜂窝粘附到所述生物传感器的变化而产生的细胞的质膜上。该协议将讨论使用RWG生物传感器通道蛋白质相互作用的检测。 RWG数据采集系统由几个部分组成。因为X-BODY Biosciences公司BIND扫描仪被用于这些实验中，我们将参照部件为这个特定的系统，它由一个平板阅读器，相关联的生物传感器，BIND扫描采集软件，和BIND查看分析软件8。光子晶体生物传感器下文提及光学生物传感器组成之一个周期性排列以电介质材料2或三个维度可防止光传播特定波长和方向。光子晶体结构是基于这种现象叫伍德的异常。首先由伍德在1902年发现，这些异常都是在光的地方在特定衍射级的强度迅速变化发生在某些窄频带光衍射光栅反射光谱观察到的效果。 1962年，和•海斯尔提出Oliner木材的异常现象的新理论在有限的时间内光栅产生一个常设的谐振波长9。在这里所描述的光学生物传感器，伍德异常被用来产生一个RWG生物传感器的，当用白光刺激仅反映非常窄的波长频带（通常介于850-855纳米）。 个体的生物传感器包括一个低折射率的塑料材料与涂有薄薄的一层高折射电介质材料的二氧化钛（TiO 2的）的周期性表面结构。当BIOSENS或者被点亮以宽广的波长的光源，光子晶体​​的光学光栅反射一个狭窄的范围内这是用分光光度计在BIND扫描仪测量的光的波长。反射的波长（峰值波长值，或PWV）的峰强度，然后从该信号来计算。的光的变化后，在折射率变化的PWV作为生物传感器表面附近内的增加或减少的质量（〜150纳米）的结果。光学生物传感器被纳入一个标准学会生物分子科学（SBS）384 - 孔微量培养板用于这些实验中使用的测定法。 RWV生物传感器被用于在添加外源的信号来检测活细胞中的变化10。细胞直接培养到RWG生物传感器的表面，并且在折射的本地索引的变化是在与特定的刺激治疗监测。质量变化在生物传感器的方向可以是决定的，因为增加从附近的生物传感器的质量增加验光结果和本地索引是按增加PWV。相反，在大规模的减少产生的PWV下降。在检测到的脉搏波速度的变化会导致从众多的细胞事件，包括改变细胞粘附，蛋白质招募/释放，细胞内吞作用和回收，胞吐作用，细胞凋亡和细胞骨架重排。例如，该测定可检测钾通道和其它细胞质或细胞骨架成分之间变化的信道 - 蛋白质相互作用的。的情况下，但是，必须发生在接近传感器的〜150nm的检测区域内，并在附属小区，靠近质膜的情况下。采集细胞反应进行与BIND软件扫描并生成每个384口井的SBS板的图像表示。该系统具有高达3.75微米/像素的分辨率，可检测单个细胞的活动，并既可以用于与细胞系（例如HEK293或CHO细胞），或与多种生理上相关的原代细胞。在BIND View软件图像分析允许个人和细胞群体中的细胞反应的检测。作为生物传感器结合到标准的SBS 384孔板，该系统易于适应高通量筛选（HTS）。 共振波长的光栅的光学生物传感器已使用过以下激活的GPCR 11来检测附近的细胞质膜质量变化。我们实验室已经克隆了钠激活（K Na）的钾通道，斯莱克-B和油滑12。两个臂-B和油滑通道的活性已被证明是非常强烈的直接激活蛋白激酶C（PKC）13的的影响。 Gαq蛋白偶联受体，如M1毒蕈碱受体和mGluR1的代谢型谷氨酸为r的活化eceptor，有力地通过PKC活化调节通道活性。这K个钠通道有助于神经细胞适应持续刺激期间和调节动作的定时的准确度电势14。松弛渠道已知与多种胞质信号分子，包括FMRP，脆性X智力迟钝蛋白15,16的相互作用。突变松弛导致婴儿期（MMPSI），癫痫的早期发作的形式导致严重的发育迟缓17多个迁移部分性发作。

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">DMEM, High Glucose</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Leibovitz&#39;s L-15 Medium</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11415-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Penicillin-Streptomycin (10,000 U/ml)</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Geneticin G-418 Sulfate</td>
			<td style="width:172px;">American Bioanalytical</td>
			<td style="width:119px;">AB05057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">HBSS</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Fibronectin from human plasma</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">F0895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Albumin from chicken egg white</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">DPBS</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Hausser Bright-Line Phase Hemocytometer</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Carbamoylcholine chloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">SFLLR-NH2 trifluoroacetate salt</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Finnpipette (5-50 &#181;l)</td>
			<td style="width:172px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">4662090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIND Scanner System</td>
			<td style="width:172px;">X-BODY Biosciences</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384-well TiO2 coated plates</td>
			<td style="width:172px;">X-BODY Biosciences</td>
			<td style="width:119px;">TiO-96-CM</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of label-free optical biosensors to detect modulation of potassium channels by g-protein coupled receptors

离子通道通过选择性地允许离子流通过质膜1控制神经元和其它可兴奋细胞类型的电性能。来调节神经细胞的兴奋性，离子通道的生物物理学性质通过信号转导蛋白和分子，这常常结合到通道本身以形成异聚复合信道2,3修改。传统的分析检查通道和调节蛋白之间的相互作用需要外源性标记，可以潜在地改变蛋白质的行为和减少靶的生理相关性，同时提供的信息很少的相互作用的时间过程中的活细胞。光学生物传感器，如X-BODY Biosciences公司的BIND扫描系统，使用了一种新的无标记技术，谐振波长光栅（RWG）的光学生物传感器，以检测共振改变反射光接近传感器。该测定法允许的相对的检测活细胞粘附于从细胞粘附配体诱导的变化和扩展性，毒性，增殖，并改变蛋白质 - 蛋白质相互作用的靠近质膜产生的生物传感器表面的底部部分内的质量变化。 RWG光学生物传感器已用于以下激活G蛋白偶联受体（GPCR），受体酪氨酸激酶，与其他细胞表面受体来检测附近的细胞的细胞膜的变化质量。在离子通道蛋白相互作用的配体诱导的变化，也可以使用该测定法的研究。在本文中，我们将描述用于检测臂-乙酸钠活化的钾（K Na）的通道由G蛋白偶联受体的调节的实验步骤。

松弛-B稳定转染的HEK-293细胞，并控制未转染的HEK-293细胞接种于1000个细胞/孔于384孔，ECM涂覆的RWG生物传感器板。从中央1.5生物传感器的毫米2的图像被记录，分辨率为3.75微米/像素（ 图1）。质量密度梯度图生成和前30分钟后加入化合物与BIND的扫描软件。 BIND的查看软件被用来确定在G蛋白偶联受体激动剂除了此消彼长PWV减去PWV后加入化合物从基线PWV。 内源?...

Watch this Scientific Journal Video about Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors at JoVE.com

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

光学生物传感器技术可检测的变化在质量近质膜在活细胞中，并允许1到跟随在这两个单个细胞和细胞群的细胞反应。这个协议将描述检测钾通道通过使用这种方法的G蛋白偶联受体在完整的细胞调制的。

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 ...

use-label-free-optical-biosensors-to-detect-modulation-potassium

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Yale School of Medicine. 光学生物传感器技术可检测的变化在质量近质膜在活细胞中，并允许1到跟随在这两个单个细胞和细胞群的细胞反应。这个协议将描述检测钾通道通过使用这种方法的G蛋白偶联受体在完整的细胞调制的。 

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

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) ...

科研

生物工程

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.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

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).

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 ...

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 ...

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.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

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.

无标记技术的单细胞分泌物的时空成像

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

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 ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip：蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...