笔者想感谢加拿大自然科学和工程研究理事会提供该项目的资金支持。

1，干海藻生物质的分离，以用作荧光读数标准<ol><li>除去从生长的藻类培养物，将提供至少200毫克干生物量的样品体积，400-600毫克，是优选的。 </li><li>在以10,000 xg 4℃10分钟离心样品。弃去上清液，并用磷酸缓冲液配制成相同的pH作为生长培养基等体积的洗涤沉淀。 </li><li>共3洗涤步骤重复步骤1.2。 </li><li>重新悬浮在去离子水中沉淀并转移到一个预先称重的称重盘。让表面干燥，50℃培养48小时。注意：在真空条件下干燥将减少干燥时间，并在温度高于50℃下干燥的培养可以使悬浮困难。 </li><li>店铺干燥藻培养物在室温下以备将来使用。 </li></ol>中性脂肪由正己烷提取物2。重力定量（来自布莱和代尔4改编） <ol><li>在称重盘尺寸约为50毫克的干藻生物量。注：大众从40-80毫克可重复性不丧失使用。 </li><li>生物质转移到研钵中预先用己烷洗涤。如果需要的话，用己烷使用巴斯德吸管，以生物质完全转移到研钵少量（1毫升）洗称量皿。注：正己烷是一种极易挥发和有毒物质。它必须在通风橱中有适当的防护衣物进行处理。 </li><li>磨藻类生物质为用杵5分钟。首先用温和的研磨，逐渐增加强度。磨生物质转化为在5分钟内一个细腻的糊状物。如果传送的生物量的砂浆时过量使用己烷，最好是等待己烷研磨之前蒸发。 </li><li>加几毫升己烷的砂浆和混合所得到的浆料用杵直到它被均化。确保所有的细胞碎片附着在铁道部的墙壁焦油被撞倒自由悬浮于液体中。 </li><li>己烷细胞量的混合物转移到离心管中。注：离心管必须是玻璃或合适的聚合物用己烷如特氟隆兼容。 </li><li>直到所有的生物质已被转移到离心管中（3 - 5倍）重复步骤2.4和2.5。 </li><li>离心机的样品在以10,000×g下4℃放置20分钟。 </li><li>小心吸取上清到一个预先称重的金属重量菜。储存在通风橱。 </li><li>加入3毫升己烷以沉淀并大力旋涡1分钟进行2 次己烷萃取。 </li><li>重复步骤2.7和2.8。如果需要的话，第二次提取过程中运行的样品进行30分钟的离心分离机，以确保所有细胞碎片完全平息。确定油重量法大规模提取正己烷后完全蒸发。 </li></ol> 3，荧光中性脂肪用尼罗红的定量（如度报告特德由德拉奥斯西格勒等 11） 注：需要荧光读数藻类悬浮在5 g / L的只有10微升。一般来说，从1.5毫升培养液的干燥藻类生物量隔离是绰绰有余。另外，在分光光度计的灯的光强度可以随时间降解。这是推荐的标准包括在每一个实验中，以确保在变化的仪器不增加不必要的错误的测量。 <ol><li>准备一个尼罗红溶液在溶解于醇试剂级乙醇10微克/ ml的浓度。在4℃下保存该解决方案在黑暗中</li><li>制备在去离子水中并存储一个30％（体积/体积）乙醇溶液在4℃下</li><li>准备所有藻类样品在相同的生物量浓度（5 g / L的建议），并用同样的方法在测量中使用的标准。由任一悬浮于适量的预先干燥的样品做这个的磷酸缓冲液（0.6克/升磷酸氢二钾，1.4 g / L的磷酸二氢钾），或测量浊度后用磷酸盐缓冲液调节生长的藻类培养物的浓度为5克/升。注意：在活的藻培养物上进行的测量通常具有与它们取决于浊度校准曲线的精确度相关的误差较大。重悬干燥的样品，可能需要使用一个均化器充分分散的生物质。 </li><li>对于每个样品，混合80微升30％的乙醇溶液中，加入10μl的尼罗红溶液，和10μl的藻类悬浮液在96孔板的单个孔。为了正确地计算荧光测定的变异，进行5次重复每个样品。 </li><li>执行两点校准曲线与之前以占一天到一天中的变化和仪器准备准备标准。使用s制备的标准进行荧光测定AME过程作为样本。注：一般两点就足够了仪器的校准，三点可以运行来验证线性度。 </li><li>执行在多板读数器分光光度计的荧光测量。以下条件被发现，得到最一致的结果11： <ol><li>摇在1,200 rpm下，轨道3mm时，持续30秒。 </li><li>孵育在40℃下10分钟。 </li><li>摇在1,200 rpm下，轨道3mm时，持续30秒。 </li><li>记录荧光，激发波长为530纳米，在604 nm发射。 </li></ol></li><li>使用从内部标准将结果转换的​​荧光测量，以油含量。 </li></ol> 4，荧光显微镜技术注：在第3条所述的染色协议被设计用于定量分析，但它也​​可以是有用的，以提供染色为基础的技术的视觉表示用于教育和说明性的普rposes。以产生样品荧光的一个的图像，需要使用光学显微镜与传统的传输和额外的落射荧光照明光源。在530纳米（绿）和604纳米（红色）范围的激发和发射光滤波器，分别被需要的尼罗红染色，以及一个安装在显微镜照相机相关联的软件。用明视场显微镜配备了摄像头和单色RGB转换器单元被收购在这项研究中（ 图1）所示的图像。使用这些工具的制造尼罗红的荧光图像的过程概述如下： <ol><li>根据协议第3条准备一个文化样本与尼罗红染色。注：在5 g / L的浓度范围内的样品产生足够的细胞密度无滑动人满为患。 </li><li>完成染色方案的步骤3.6之后，按照标准实验室方法制备经处理的样品的显微镜载玻片上。 </li><li>在传输模式的显微镜开始，装载准备滑入显微镜，并找到细胞所需的放大倍率（本文中显示的图像被收购与100X目标）。 </li><li>一旦集中，从传输切换显微镜落射荧光照明模式。光源现在应该直接从物镜来（这可以在视觉上通过观察滑和物镜之间的空间中得到证实）。 </li><li>插入一个绿色激发滤光片进入光源和红色发射滤波器的观察光路中;染色细胞的荧光现在应该直接可见通过目镜。 </li><li>从目镜切换显微镜观察模式下的安装照相机和使用浏览软件来捕获荧光的细胞的图像。根据相机的灵敏度，样品最初可能不会出现在预览窗口（ 即屏幕上会是黑色的）;以解决这个问题，调整曝光时间和照相机增益到一定水平，所述细胞是可见的。具体的设置将与仪器，设备，和细胞类型而有所不同。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在标准曲线所用的藻类必须是那些被测量相同的实验条件下培养相同的物种。显著改变培养基组成，栽培技术，和染色的协议会影响读数的荧光的强度。己烷萃取（在第1和2中所述）被用于确定在标准曲线用样品的中性脂质含量。为准确的荧光强度测量结果，所有的样品必须在相同的生物量浓度（5 g / L的是在本研究中所用）进行分析。含油量然后可以通过比较，通过标准曲线来容易地确定。对于?...

由于其存储在一定应力条件下脂质体的能力，藻类已经收到了极大的关注，近年来作为一种潜在的可再生燃料来源1,2。中性脂肪可以在适当的生长条件3占细胞干重的60％以上。然而，金融业并没有一个简单，干净，快速，可靠的标准化协议，以定量藻细胞的脂含量，以适当监测生​​物过程的性能，分析文化和屏幕的新品种。 该布莱-代尔重量法开发了一些50年前之间保持4,5今天最常用的技术。虽然这个过程很简单，可靠，方便外出携带，它是耗时的，就必须大样本量，并使得有毒溶剂的使用。这是不实际的，用于分析许多样品从发酵跑步或筛选新的石油资源丰富的菌株。其他方法有BEEN发达，但通常需要先进的设备和不规范6。 这已经获得了极大的兴趣另一种是尼罗红染色。尼罗红，发荧光优先在非极性环境中的染料，已被用于确定或量化脂质体在各种生物体，包括线虫7，酵母8，细菌9和藻类10-19。涉及尼罗红初始技术大多是定性或半定量的，色斑与单反应杯光度法或流式细胞术结合起来。此外，一些类藻类如绿藻有厚胞孔的大多是不可渗透的染料，这限制了该技术10的范围内。 在尼罗红染色法的最新改进，据报道，绕过协议10,11的初始缺点。在卡尔的存在下细胞染色IER溶剂如DMSO中10或乙醇10,11线性化油含量和吸收率之间的关系，从而允许可靠的定量测量。溶剂可帮助通透细胞膜，使尼罗红分子能够通过。此外，结合分光光度计与微孔板读数功能使适合于定量分析的高通量协议。 在这篇文章中我们详细介绍通过染色文化与尼罗红在乙醇，温和溶剂存在测量藻细胞的含油量的简单方法。为了最准确地解释在测量背景噪声，标准曲线相关联的荧光强度，含油量是使用已知的石油成分的藻细胞开发。该方法是改编自先前公布的协议10,11。通过使用96孔分光光度计，1是能够分析在一小时THA样品相同量的T公司将需要数天由重量法来监测。此外，通过使用所需的藻类物种的代表性样品校准该方法中产生相对精确的测量可直接解释的。存在着许多协议概述藻类染色用尼罗红为不同的菌株和应用进行了优化的方法;这里介绍的协议最初是由德拉奥斯西格勒等 11 Auxenochlorella protothecoides，小球藻 ， 栅藻二形 ，和斜生栅藻 ，虽然它很可能适用于许多种类和类。它的设计与监测生物过程性能的具体应用，它同样适用于预先干燥试样和湿样品从不断增长的文化。

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			<td height="21" style="height:21px;width:347px;">Dry Weight</td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">25 ml disposable pipettes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">13-676-10K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipette Bulb</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">13-681-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">40 ml Nalgene Teflon Centrifuge Tubes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">05-562-16A</td>
			<td>Teflon needed for hexane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Weigh Dishes (polypropylene)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">2-202B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1.5 ml micro-centrifuge tubes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Centrifuge</td>
			<td style="width:152px;">Sorvall</td>
			<td style="width:152px;">RC6plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Drying Oven (Fisher 625D)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">13-254-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Storage vials</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">0337-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bench-top microcentrifuge (Eppendorf 5415D)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">05-40-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Gravimetric Quantification</td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Porcelain Mortar (Coorstek)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">12-961A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Porcelain Pestle (Coorstek)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">12-961-5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">40 ml Centrifugation tubes (FEP)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">05-562-16A</td>
			<td>Could also use glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pasteur Glass Pipettes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">13-678-20C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Aluminum weigh dishes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">08-732-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Hexanes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">H292-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:347px;">Fluorometric quantification of oil content</td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Fluorescence multi-well plate reader</td>
			<td style="width:152px;">Thermo Lab Systems</td>
			<td style="width:152px;">Fluoroskan Ascent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fluorescence reader software</td>
			<td style="width:152px;">Thermo Lab Systems</td>
			<td style="width:152px;">Ascent Software 2.6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">COSTAR 96 well plate with round bottom</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">06-443-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Nile Red&#160;</td>
			<td style="width:152px;">Sigma</td>
			<td style="width:152px;">N3013-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ethanol (Alcohol reagent grade)</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">AC65109-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;"></td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Imaging Fluorescent cells</td>
			<td style="width:152px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Leica DMRXA2 (or equivalent) microscope</td>
			<td style="width:152px;">Leica</td>
			<td style="width:152px;">DMRXA2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microscope slides</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microscope cover slips</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:152px;">12-541B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Camera</td>
			<td style="width:152px;">Qimaging</td>
			<td style="width:152px;">Retiga Ex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Imaging software</td>
			<td style="width:152px;">Qimaging</td>
			<td style="width:152px;">QCapture v.1.1.8</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol for measuring neutral lipids in algal cells using fluorescence

藻类被认为是优秀的候选人，由于其天然脂质存储功能的可再生燃料来源。强有力的监测藻类发酵过程和筛选新的石油资源丰富的菌株需要一个快速，可靠的协议，用于测定细胞内脂质含量。目前的做法主要依靠重力方法来确定油含量，技术开发几十年前是耗时且需要大量的样本量。在本文中，尼罗红，已经被用来确定脂质体在许多类型的有机体的存在下的荧光染料，掺入一种简单，快速和可靠的协议，用于测量Auxenochlorella protothecoides的中性脂质含量，绿色藻类。该方法使用乙醇，相对温和的溶剂，通透染色前的细胞膜和一个96孔微板中的荧光强度测量，以增加样品的容量。它已经设计编带的监测生物过程性能的具体应用。预先干燥的样品或活的样品从生长培养可在测定中使用。

沾上尼罗红染料代表藻细胞如图1所示。部分A和图1显示A的图像乙 protothecoides生长在过量的氮，从而导致非常低的细胞内脂质蓄积。在部分C和D，样品A的在氮气氛下限制生长protothecoides示。根据透射照明，细胞的脂质体，可以可视化通过仔细检查图1C中 ，在那里它们将显示为有光泽?...

Watch this Scientific Journal Video about A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence at JoVE.com

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

一个简单的协议采用尼罗红染色法来确定藻细胞中的中性脂质含量进行说明。这个节省时间的技术提供了一种替代传统的重量为基础的脂质定量协议。它被设计用于监测生物过程性能的具体应用。

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

a-simple-rapid-protocol-for-measuring-neutral-lipids-algal-cells

Research

JoVE Journal

Chemistry

35.3K 次观看.  University of Alberta. 一个简单的协议采用尼罗红染色法来确定藻细胞中的中性脂质含量进行说明。这个节省时间的技术提供了一种替代传统的重量为基础的脂质定量协议。它被设计用于监测生物过程性能的具体应用。 

视频: A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

功能性降冰片烯的催化聚合插入一种简单高效的协议

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

使用有机锂试剂的一个协议安全的锂化反应

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

快速超临界萃取制备催化气凝胶的制备与试验研究

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer by generating a charged structure provides an opportunity to use extraction as a simple purification technique. By combining the use of a miscible organic solvent with saturated sodium bisulfite, aldehydes and reactive ketones can be successfully transformed into charged bisulfite adducts that can then be separated from other organic components of a mixture by the introduction of an immiscible organic layer. Here, we describe a simple protocol for the removal of aldehydes, including sterically-hindered neopentyl aldehydes and some ketones, from chemical mixtures. Ketones can be separated if they are sterically unhindered cyclic or methyl ketones. For aliphatic aldehydes and ketones, dimethylformamide is used as the miscible solvent to improve removal rates. The bisulfite addition reaction can be reversed by basification of the aqueous layer, allowing for the re-isolation of the reactive carbonyl component of a mixture.

Here, we present a protocol to remove aldehydes and reactive ketones from mixtures by a liquid-liquid extraction protocol directly with saturated sodium bisulfite in a miscible solvent. This combined protocol is rapid and facile to perform. The aldehyde or ketone can be re-isolated by the basification of the aqueous layer.

用亚硫酸氢萃取协议分离混合物中的醛和反应性酮

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...