Name: automated 90sr separation and preconcentration in a lab-on-valve system at ppq level
Uploaded: 2018-06-06T14:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level at JoVE.com

作者希望 aknowledge 参与项目不同步骤的同事和人员。在 JRC 卡尔斯鲁厄设计办公室的同事, Knoche 和 Volkmar 欧内斯特, 在 JRC 卡尔斯鲁厄讲习班基督教 Diebold 和 Küst 为实验室的设计和制造阀门 (爱) 歧管和林瓦尔德 GmbH 为停止流检测模式技术的软件实现。

注: 使用设计用于超痕量分析的设备, 采用高纯度水 (18.2 MΩ cm) 制备解决方案。用石英亚沸腾蒸馏装置提纯硝酸。净水系统和次沸蒸馏装置均在洁净室操作。
警告: 90Sr 具有强烈的毒性和致癌性。在进行实验时, 应采取适当的安全措施, 包括工程控制系统和个人防护设备。
1. 实验准备
注意: 有关软件体系结构及其功能的详细说明可在其他地方找到23。
<ol><li>将模块化阀位置出口端口连接到无线电流量检测器的入口端口。</li>
	<li>将触发线连接到选定的探测器。</li>
	<li>确保爱的端口已正确连接。确保为整个协议提供足够数量的工作解决方案, 并且液体取样油管仍将被淹没。</li>
	<li>确保 Autosampler, 启动 Autosampler 软件, 并通过软件接口初始化 Autosampler。单击初始化按钮以生成设备和 PC 之间的通信。</li>
	<li>确保 AutoRAD 软件已启动, 请使用 "选项" 选项卡检查通信端口, 并通过用户界面初始化软件。有关软件图形界面的详细信息, 请参阅讨论部分。</li>
	<li>确保检测软件中的分析序列被编程。通过输入所需的步骤数以及每个设备的任务和速度, 使用 AutoRAD 软件中的方法编辑器选项卡程序视图序列。 注意: 在以前的发布23中可以找到软件操作的详细说明。</li>
	<li>请咨询本地防护人员, 并使用90Sr 为整个过程采用所需的防护测量。</li>
</ol>2. 系统清洗
<ol><li>在 90 mL·min-1上, 从烧瓶中装载10毫升 18.2 MΩ厘米的水。确保将注射器阀位置设置为此步骤的 in 位置. 在这个位置, 加载发生直接到注射器, 而不是通过爱。</li>
	<li>在 90 mL·min-1的流速下, 通过保持线圈将水丢弃到废料中。</li>
	<li>在 3 mL·min-1的流速下, 将3毫升乙醇装载到保温线圈上。将注射器阀位置设置为出。</li>
	<li>将乙醇以 3 mL·min-1的流速降低到探测器线圈。</li>
</ol>3. 对爱的负载树脂
<ol><li>搅拌树脂悬浮在水中 (12 mg·mL-1), 通过爱提取3毫升。将流设置为 3 mL·min-1
</li>
	<li>以 1.2 mL·min-1的流速将树脂悬浮液滴入柱形通道
</li>
	<li>清洗保温线圈, 将树脂残留物放到柱形通道上。为此, 负载9毫升 18.2 MΩ厘米水从烧瓶到注射器的流速为 90 mL·min-1。确保注射器阀位置位于 in 位置. 在 3 mL·min-1的流速下, 通过保持线圈将水丢弃到废料中。</li>
</ol>4. 分析序列
<ol><li>列调节<ol>
<li>将2毫升的 HNO3 (4 米) 通过爱的持有线圈加载。将流设置为 6 mL·min-1。</li>
		<li>将 HNO3拖放到列上, 其流速率为 1.2 mL·min-1。</li>
	</ol>
</li>
	<li>采样的加载和消除干扰<ol>
<li>从 autosampler 到持有线圈的负载样品 (1.3 毫升) 的流速为 6 mL·min-1。</li>
		<li>以 1.2 mL·min-1的流速将示例拖到列上。</li>
		<li>将0.5 毫升的 HNO3 (4 M) 加载到持有线圈, 其流速为 6 mL·min-1。</li>
		<li>用0.5 毫升的 HNO3 (4 米) 冲洗该列, 以洗脱矩阵干扰的速率为 1.2 mL·min-1.</li>
	</ol>
</li>
	<li>样品的洗脱和测量<ol>
<li>将5毫升 18.2 MΩ厘米的水装载到保持线圈上, 流速为 6 mL·min-1。</li>
		<li>触发探测器。将闪烁液体的流速设置为检测器软件中的 2 mL·min-1 。定住到十年代。样品线圈的体积为2毫升。</li>
		<li>冲洗列。</li>
	</ol>
</li>
	<li>取样探头和夹持线圈的冲洗<ol>
<li>将0.6 毫升的 HNO3 (1%) 加载到持有线圈, 其流速为 6 mL·min-1。</li>
		<li>以 6 mL·min-1的流速将0.6 毫升的空气加载到保持线圈上。</li>
		<li>将混合物的1.2 毫升排出废料。</li>
	</ol>
</li>
	<li>树脂置换<ol>
<li>在 3 mL·min-1的流速下, 将0.2 毫升乙醇装载到保温线圈上。</li>
		<li>用0.2 毫升乙醇冲洗柱, 流速为 1.2 mL·min-1。</li>
		<li>用0.5 毫升水冲洗柱通道, 流速为 0.45 mL·min-1。</li>
		<li>将使用过的树脂排出废料。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

放射性特征和保护是核设施生命周期所有阶段的关键问题。核设施退役期间需要进行放射性核素测定, 需要不断改进分析程序。这需要提高选择性和灵敏度, 缩短分析时间。AUTORAD 原型满足了这些要求。此外, 该原型是便携的, 并允许现场确定。建议的自动在线方法成功地应用于测定水样品中的90Sr 活动。
图 1显示了 AutoRAD 系统的示意图。使用商用图形编程语言 LabVIEW 2014 实现了与原型的通信。部署了用于配置、编程和控制接口的虚拟仪器软件体系结构 (VISA) 模式。图 2显示了图形用户界面屏幕, 在系统初始化之前可以验证端口配置。签证模式在操作系统和程序环境上是独立的, 因此提供了特殊的通用性。已开发的软件的基本结构和特点在以前的出版物24中得到了广泛的讨论。通过使用 RS232 接口控制 PC 机的滴定管, 实现了改进, 从而允许同时加强对两滴定管的控制, 以及在停止流模式下进行测量的可能性。相反, 当系统背压增加时, RS232 连接会迅速反应。这可能导致系统错误和测量停止。因此, 在树脂的加载过程中, 必须特别注意样品的离子强度。
用锶稳定同位素86sr 作为放射性90sr 的代理, 并将 AUTORAD 系统耦合到 ICP-MS 而不是 beta detctcor, 实现了实验条件的优化。系统。图 3显示了 ICP-MS 86Sr 洗脱配置文件。获得的86Sr 洗脱配置文件与以前报告的总锶使用低压分离设备25的结果完全一致, 并已被减去空白。采用最小二乘法线性回归法生成不引入额外不确定分量的目的曲线拟合。所得到的线性度为 0.995, 与p值小于意义水平。2 pg·g-1的 LOD 是根据柯里26对空白的重复测量确定的。该方法的重现性基于峰值面积的相对标准偏差, 根据三次重复运行计算, 在10到 120 pg·g-1的范围内始终小于4%。在洗脱剖面中目击的肩部最有可能是由于自动 AUTORAD 配置中列的非最佳包装造成的。
图 4显示了使用无线电流量检测器的90Sr 配置文件。AUTORAD 系统能够有效地在水样品中分离90Sr。
图 5显示了90Sr 浓度与信号在感兴趣范围内是线性的依赖性。导出的检测限制在成品中。g-1范围, 启用, 不作进一步修改, 在核退役和废物表征样品中确定放射性锶。该方法的重现性, 利用流闪烁探测器, 并根据峰值面积的相对标准偏差, 在研究的浓度范围内约为30%。然而, 目前的原型设置限制了它在环境样品中的应用, 这主要是由于探测器的时间数很短。此外, 复杂的矩阵可能饱和的树脂在迷你柱。
在测量过程中, 哈密顿和无线电流量检测器泵停止流动的技术将被实现。这项功能将延长停留时间在样品区域的最大的部分在流动细胞之内, 提高灵敏度。因此, 在样本退出检测器之前, acculated 有统计学意义的信号。这种方法将改进计数统计和检测限制。此外, 正在开发一个新的应用, 其中包括一个额外的离子交换小柱, 用于去除可能影响复杂基质样品中不同放射性核素的保留和分离的基质元件。

大约150个商业核电厂正在退役, 但如果将研究和再加工设施考虑到1, 核设施的总数量就会大得多。核设施退役非常昂贵, 涉及运输和关闭污染材料的现场测量。通过采用现场和灵活的测量技术1可以节省开支。因此, 迫切需要迅速提供支持旧核设施退役的现场分析方法。伽玛发射器可以很容易和有选择地确定使用伽玛光谱学, 但在现场分析的方法, 难以测量 (HTM) 放射性核素2。在 HTM 中, 90Sr 由于其毒性和高能量排放引起了极大的兴趣。它的决心是耗时的, 需要分离从干扰和量化使用或分光光度法3,4,5,6,7, 8,9,10,11,12,13。
标准的放射化学方法费时费力, 通常需要重复才能获得足够的锶产量。因此, 迫切需要更快、更准确的方法。除标准分离协议外, 流动注射技术还适用于放射性同位素5、14、15的纯化和/或预浓缩。实验室阀门 (爱) 装置是流动技术的进一步发展。它们是可编程的、基于流的平台, 具有不同的耦合模式, 并且具有高通用性16。这种装置允许在检测之前自动分离和预浓缩分析器, 从而增加再现性和重复性8,17,18,19。与 multisyringe 流泵耦合的爱系统被广泛用于检测放射性核素, 因为试剂消耗和废物产生的最小化8,10,17,18,19,20. 然而, 关于超微量水平在线检测的报告研究是稀疏817。
放射性材料的现场测量有许多优点和优点, 但在90Sr 监视中没有应用流闪烁。基本上, 量化是使用在线检测器, 混合的洗脱液来自爱的设备与闪烁鸡尾酒11, 21, 22.然后通过计数单元泵浦混合物, 用配对光电倍增管进行测量。由于单元格体积小, 测量时间为秒刻度。
本研究的目的是开发一种全自动的在线锶检测方法, 其范围广泛, 涵盖了与环境有关的浓度, 也包括在核工业废料流中发现的。该平台是移动的, 可以安装在车辆上进行水样品的现场分析。

<table><tbody><tr><td>microLAB 600 series</td><td>Hamilton</td><td>ML600EE6910</td><td>Two dual syringe pump system, equiped with two 10 mL glass syringes. The instruments are interconnected using the CAN port (daisy chain).</td></tr><tr><td>FlowLogic U</td><td>LabLogic</td><td>SG-BXX-05</td><td>Liquid scintillator with high flash point</td></tr><tr><td>&amp;szlig;-RAM 5</td><td>LabLogic</td><td></td><td>flow detector, 2000 &amp;mu;L coiled Teflon flow cell.Software Laura 4.2.8 (LabLogic, England) run on desktop PC and connected to the detector via USB</td></tr><tr><td>SC-&amp;mu; DX Autosampler</td><td>Elemental Scientific Instruments (ESI)</td><td></td><td></td></tr><tr><td>Cheminert selector</td><td>Valco Instruments Co. Inc.</td><td></td><td>in-house made Lab-on-Valve has been mounted on this selector</td></tr><tr><td>Modular Valve&lt;br/&gt; Positioner (MVP)</td><td>Hamilton</td><td></td><td></td></tr><tr><td>mini magnetic stirrer</td><td>IKA</td><td></td><td></td></tr><tr><td>Nitric Acid Suprapur 65%</td><td>Merck</td><td>1.00441.1000</td><td>purified using quartz sub-boiling distillation unit</td></tr><tr><td>Sr-resin</td><td>Eichchrom Tecnologies, Inc</td><td>SR-B100-A</td><td>particle size 100-150 &amp;micro;m</td></tr><tr><td>Water system Elix 3 in combination with Mili-Q Element A10</td><td>Millipore</td><td></td><td>high-purity water (18.2 M&amp;Omega; cm)</td></tr><tr><td>Sr-90 standard</td><td>Eckert &amp;amp; Ziegler</td><td>7090</td><td>Sr-90 concentration 1.915 kBq/g &amp;plusmn; 3.0%, reference date 15-May-2016 12:00 PST</td></tr><tr><td>MLS quartz sub-boiling distillation unit</td><td>MLS GmbH</td><td></td><td>Subboiling unit for the purification of HCl and nitric acid</td></tr></tbody></table>

automated 90sr separation and preconcentration in a lab-on-valve system at ppq level

开发了一种快速、自动化、便携的水样品中放射性锶的分离和测定系统, 采用 Sr 树脂和多序贯流注入分析法。放射性锶的浓度由流动闪烁计数确定, 允许在线和现场确定。该系统可在工业相关水平上确定放射性锶, 而无需进一步修改, 使用的总分析时间为每水样品少于10分钟。检测的限制为 320 fg·g-1 (1.7)。

开发并实现了由自制的基于 LabVIEW 的软件操作的全自动 AUTORAD 平台 (图 1)。该软件允许在用户友好的环境中为日常操作提供足够的灵活性 (图 2)。通过将该平台与不同的检测器 (图 3) 耦合在一起, 也证明了该系统的通用性。该方法的适用性已在含90Sr 标准 (图 4) 的水样中显示。对参数的线性度、线性范围、检测极限 (LOD) 和重复性进行了评估 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57722/57722fig1.jpg"> 图1。显示端口配置和使用试剂的 AutoRAD 系统的示意图表示。中心端口通过保持线圈 (10 毫升) 连接到注射器泵;在房子里制造了甲基丙烯酸酯。<a href="/files/ftp_upload/57722/57722fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57722/57722fig2.jpg"> 图2。AutoRAD 软件选项菜单.右下角区域显示分配的端口。通过 "向下滚动" 菜单, 用户可以在分配中进行更改。<a href="/files/ftp_upload/57722/57722fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57722/57722fig3.jpg"> 图3。86采用电感耦合等离子体质谱 (ICP-MS) 作为探测器的 Sr 洗脱峰.在洗脱的第一个100s 中, 锶从柱体中洗脱定量。<a href="/files/ftp_upload/57722/57722fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57722/57722fig4.jpg"> 图4。34.5 @ 1 90Sr (6.6 pg·g-1) 洗脱峰使用ß RAM 5 作为探测器.居住时间在卷是四十年代。建议的90Sr 方法的总体恢复率为 70% 5%。<a href="/files/ftp_upload/57722/57722fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57722/57722fig5.jpg"> 图5。计数率与放射性锶浓度的校准曲线。获得了良好的线性度 (R2 = 0.997)。检测限制被计算为 320 fg·g-1 5 (1.7), 由三点三乘以空白的标准偏差。<a href="/files/ftp_upload/57722/57722fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level at JoVE.com

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Ppq 级实验室系统中的自动90Sr 分离和预富集

在这里, 我们提出了便携式 autoRAD 平台快速辐射分离和确定的90Sr, 一个重要的裂变产品高度相关的核废料。

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

automated-90sr-separation-preconcentration-lab-on-valve-system-at-ppq

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Chemistry

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Atoms and Elements

SCIENTISTS IN ACTION

7.9K 次观看.  European Commission, DG Joint Research Centre - JRC. 在这里, 我们提出了便携式 autoRAD 平台快速辐射分离和确定的90Sr, 一个重要的裂变产品高度相关的核废料。

视频: Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

科研

生物化学

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

化学

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

癌症研究

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

生物工程

Preparing an Isotopically Pure 229Th Ion Beam for Studies of 229mTh

A methodology is described to generate an isotopically pure 229Th ion beam in the 2+ and 3+ charge states. This ion beam enables one to investigate the low-lying isomeric first excited state of 229Th at an excitation energy of about 7.8(5) eV and a radiative lifetime of up to 104 seconds. The presented method allowed for a first direct identification of the decay of the thorium isomer, laying the foundations to study its decay properties as prerequisite for an optical control of this nuclear transition. High energy 229Th ions are produced in the &#945;&#160;decay of a radioactive 233U source. The ions are thermalized in a buffer-gas stopping cell, extracted and subsequently an ion beam is formed. This ion beam is mass purified by a quadrupole-mass separator to generate a pure ion beam. In order to detect the isomeric decay, the ions are collected on the surface of a micro-channel plate detector, where electrons, as emitted in the internal conversion decay of the isomeric state, are observed.

Preparing an Isotopically Pure 229Th Ion Beam for Studies of 229mTh

We present a protocol for the generation of an isotopically purified low-energy 229Th ion beam from a 233U source. This ion beam is used for the direct detection of the 229mTh ground-state decay via the internal-conversion decay channel. We also measure the internal conversion lifetime of 229mTh as well.

为229mTh的研究准备一个同位素纯229Th Ion光束

A methodology is described to generate an isotopically pure 229Th ion beam in the 2+ and 3+ charge states. This ion beam enables one to investigate the ...

工程学

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

[11C_SNAP-7941] 自动合成和实时动力学评估的技术方面

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

生物学

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Ppq 级实验室系统中的自动⁹⁰Sr 分离和预富集

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