This work is supported by the European Commission's 7th Framework Programme under Grant Agreement Number 287382. SY is a beneficiary of a European Commission's 7th Framework Programme Grant Agreement Number 287382. IK is a beneficiary of a FIRST doctoral school grant from the Fondazione Edmund Mach. For his work at University of Foggia, VC is supported by the Apulian Region in the framework of 'Future In Research' program (practice code 9OJ4W81).

该议定书遵循我们的人类研究伦理制度委员会的指导方针。 样品制备和自动进样器条件<ol><li> 在线生物过程监测:检测乳酸发酵过程中释放的挥发性有机酸 注意:本协议的这一部分代表了Benozzi 等报道的程序的一部分。 32 <ol><li>向每个小瓶（装有PTFE /硅胶隔片的20mL玻璃小瓶）中加入5mL巴氏灭菌牛奶。注意使用的牛奶类型，并将样品快速加热至45°C。将其转移到配有温度控制托盘（45°C）的多功能GC自动进样器。 </li><li>使用自动进样器的机器人手臂接种微生物发酵剂培养物（根据起始培养基生产商的规格）的小瓶。根据所需酸奶的类型和规格设定孵育时间由起始文化制造商报告。设置自动进样器以方便地分析一个样品，在酸奶制备过程中获得乳酸发酵的在线VOC监测。 </li></ol></li><li> 大规模筛选:监测与不同苹果基因型相关的VOC 注意:本协议的这一部分代表Farneti 等人报告的一部分程序。 33，36 <ol><li>在成熟/保存期望的阶段， 例如苹果（ 例如在商业收获阶段）。选择至少五个均匀的水果，每个克隆没有任何可见的损伤。将苹果在室温（25°C）或冷藏（4°C）下保存所需时间。 </li><li>用肉片采样器从每个苹果收集五个圆柱形盘（直径1.7厘米，厚1厘米）。包括一部分皮质组织，避免核心部分与种子。立即使样品均质并在液氮中冷冻。储存于-80°C直到分析。 </li><li>在分析前，将来自每个生物复制品的2.5g苹果样品的三次重复放入小瓶（装有PTFE /硅胶隔片的20mL玻璃小瓶）中。将样品与2.5mL去离子水，1g氯化钠，12.5mg抗坏血酸和12.5mg柠檬酸混合，并将样品保持在4℃直到分析（最多3天）。 </li><li>在40℃下孵育样品，然后设置自动进样器以自动分析VOC。 </li></ol></li><li> 空气分析:研究饮酒过程中VOC的深度释放 注意:该协议的这一部分代表Romano 等报道的程序的一部分。 28 <ol><li>从磨碎的咖啡样品中准备冲泡的咖啡。 <ol><li>使用咖啡机:报告水/粉比，所用矿泉水的种类，咖啡机的类型以及获得咖啡饮料所采用的程序（数量是咖啡机尺寸的函数）。 </li><li>使用六杯咖啡机，意大利知名的"moka"，使用450毫升水和30克咖啡粉。将煮过的咖啡放入容器中，并将其转移到恒温水浴（60°C）中。 </li></ol></li><li>对于每个咖啡冲泡，将7.5mL等分试样转移到具有塑料盖的聚苯乙烯杯（40mL）中。每个小组成员按照协议对饮料进行尝试:i）30秒的自由呼吸，ii）单次喝咖啡，然后快速吞咽，iii）3分钟的呼吸进入符合人体工程学的玻璃鼻梁28 。 </li><li>每天重复整个实验连续三天，随机抽取咖啡样本和小组成员的次序。 </li><li>通过将硅橡胶中的一次性符合人体工程学的喷嘴安装在小组成员的鼻子上进行采样。连接n通过仅在与面板主体接触的第一部分中不加热的PEEK管到PTR-ToF-MS，然后在110℃下在入口软管中加热，该入口软管将采样接口与PTR-MS连接仪器。 注意:在表2中，用Benozzi 等人报道的类似程序分析的产品列表。 32 ，Farneti et al。 33,36和Romano 等人 28报道。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 食物矩阵 </td><td> 样品数量和种类 </td><td> 参考 </td></tr><tr><td>苹果</td><td>作者筛选了由新旧苹果品种组成的190个种质资源</td><td> 33 </td></tr><tr><td>酸奶</td><td>在酸乳酪乳酸发酵过程中释放的VOCs分析了四个起动器（A，FD-DVS YF-L812 Yo-Flex，Chr.Hansen; B，FD-DVS YC-380Yo-Flex，Chr.Hansen; C，FD -DVS YC-X11 Yo-Flex，Hansen; D，YO-MIX 883，Danisco） </td><td> 32 </td></tr><tr><td>咖啡</td><td>使用从单一纯阿拉比卡咖啡混合物获得的三种不同种类的研磨咖啡:中烤，黑烤和脱咖啡因培养基</td><td> 28 </td></tr></tbody></table> 表2:分析产品清单。通过与Benozzi 等人报道的类似程序分析的产品清单。 32 ，Farneti et al。 33,36和Romano 等人 28 2.实验设计与实践预防<ol><li>执行至少三个日间生物复制品每个都有三个技术重复，每个实验模式。 </li><li>在进行样品孵育和分析之前，每个小瓶以200sccm的清洁空气冲洗顶空1分钟。 </li><li>为每个实验模式准备一个空白，在相同的样品条件下孵育和分析空白。 </li><li>随机抽样/空白的顺序进行分析。 </li><li>与用于检测VOC的其他方法类似，在使用仪器之前，限制使用香水的个人护理产品以及口香糖和香烟。在实验室中紧密封住挥发性化学物质，并在测试期间尽可能多地控制气流。 </li></ol> 3. PTR-MS仪器优化与分析注意:工具条件在参考文献中有描述（ 例如 Makhoul 等人 27 ）。 <ol><li>用商品进行样品的顶空测量以PTR-ToF-MS装置为标准配置模式。 </li><li>直接将空气注入PTR-MS漂移管顶空，无需任何处理。样品空气通过PTR-MS连续流动，因此通过简单地将PTR-MS入口的末端插入样品顶部空间来实现注射。 </li><li>设置并不断验证漂移管中的以下电离条件:110°C漂移管温度，2.30 mbar漂移压力，550 V漂移电压。这导致约140Td（1Td = 10 -17cm 2 V - 1s - 1 ）的E / N比。入口管线由在110℃加热的PEEK毛细管（内径0.04英寸）组成。默认情况下，将入口流量设置为40sccm。 </li><li>将ToF采集通道的采样时间设置为0.1 ns，对于m / z = 400的质谱，共有35万个通道。每个单个频谱是大约28,600次采集的总和持续35每个μs，导致1秒的时间分辨率。 注意:然后连续存储光谱。光谱测量信号在几秒钟内（从入口管线中更换气体所需的时间）从背景水平增长到稳定值，并且仅在进一步分析后考虑采集的光谱。 </li></ol> 4.定制数据分析注意:使用MATLAB中的过程开发了量身定制的数据分析。 <ol><li>通过基于泊松统计的方法校正离子检测器死区时间造成的计数损失，如Cappellin 等人所述。 10 。 </li><li>根据Cappellin 等人描述的程序执行内部校准38达到良好的质量精度（高达0.001 Th）。 </li><li>进行复合注释比较获得的光谱数据与参考标准的碎片数据和数据r在科学文献中报道。 </li><li>根据Cappellin 等人进行降噪，基线去除和峰强度提取。 39 ，使用修改的高斯拟合峰。 </li><li>通过Lindinger 等人描述的公式计算峰值强度，单位为ppbv（以体积计） 。 5 ，当底层化合物未知时，使用适当的反应速率系数或反应速率系数（k = 2.10-9cm 3 s -1 ）的常数值。后者引入高达30％的系统误差，如果实际系数已知40则可以解释 。 </li><li>通过执行主成分分析，方差分析，Tukey的事后检验以及适应使用R开发的现有包装的其他统计测试/分析来挖掘数据（ 例如 Cappellin 等人 </ol>

The authors have nothing to disclose.

耦合飞行时间（ToF）质谱分析仪的质子转移反应 - 质谱（PTR-MS）代表了对挥发性有机化合物的鉴定和定量的需要以及快速分析分析的必要性之间的有效折中。表征ToF质量分析仪的高质量分辨率为相关灵敏度和质谱提供了相当大的信息内容。此外，PTR-ToF-MS与自动采样器的配合以及增加自动化程度的定制数据分析工具的应用，增强了这一技术的潜力。 在许多研究和技术领域，挥?...

直接注入质谱（DIMS）技术代表了一类分析仪器方法，具有高灵敏度和鲁棒性的相当大的质量和时间分辨率，可以快速检测和定量挥发性有机化合物（VOC） 1 。这些工具方法包括MS-e-noses，大气压力化学电离质谱（APCI-MS），质子转移反应质谱（PTR-MS）和选定离子流管质谱（ SIFT-MS） 1 。每种方法的优点和缺点取决于:样品注入的种类，前体离子的来源和控制，电离过程的控制以及质量分析仪1,2 。 质子转移反应质谱（PTR-MS）是二十多年前开发的，用于实时监测和检测在空气中的最低挥发性有机化合物（VOC）的低检测限（通常为几ppbv，体积百分之十）。目前PTR-MS的应用范围从医疗应用，食品控制到环境研究5,6 。该技术的主要特点是:快速连续测量的可能性，前体离子的强烈和纯净的来源，以及控制电离条件（压力，温度和漂移电压）的可能性。这些功能允许将多功能用途与高标准化组合1,4 。事实上，该方法是基于水合氢离子（H 3 O + ）的反应，其在大多数挥发性化合物（特别是以质子亲和力高于水的特征）中诱导非解离质子转移，质子化中性化合物（M）根据反应:H 3 O + + M→H 2 O + MH + 。与其他技术相比， 例如 APCI-MS，前体离子产生和样品电离分成两个不同的仪器室（ 图1中给出了PTR-MS仪器的示意图 ）。中空阴极离子源中的水蒸汽放电产生一束水合氢离子。在此阶段之后，离子穿过漂移管，其中VOC的电离发生7 。离子然后进入脉冲提取部分并加速到TOF部分。通过飞行时间，可以确定离子8的质荷比。每个提取脉冲导致所选m / z范围的完整质谱8 。离子光谱由快速数据采集系统7记录。一个完整的频谱是典型的在一秒钟内获得，尽管可以根据信号噪声水平实现更高的时间分辨率，并且即使没有校准，也可以提供VOC顶空浓度的定量估计9,10 。 <img alt="图1" src="/files/ftp_upload/54075/54075fig1.jpg" /> 图1: PTR-MS的示意图。 PTR-MS仪器的示意图。 HC:具有中空阴极的外部离子源; SD:源漂移;六，文丘里型入口; EM，电子倍增器; FC1-2，流量控制器。转载自Boschetti 等人的许可7 。 <a href="http://ecsource.jove.com/files/ftp_upload/54075/54075fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 11 。 PTR对环境，大气，食品，技术，医学和生物科学十分有兴趣12 。 与食品基质相关的挥发性有机化合物在食品科学和技术方面具有突出的兴趣，因为它们在与气味和风味感知相关的生物现象的分子基础中起重要作用，因此在食品接受度方面。因此，我们对VOC的实时和非侵入性检测的兴趣主要涉及食品的感官品质。此外，如果我们考虑通过释放的VOC 13检测腐败和致病微生物的可能性，和/或监测挥发性有机化合物作为标记物机翼技术过程（ 例如热处理中的美拉德副产品） 14 ，VOC识别和定量是食品质量管理中感兴趣的领域。几种近来PTR-MS技术用于快速监测和定量食品基质中VOCs的应用证明了这些分析方法的广泛应用（ 表1 ）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 食物矩阵 </td><td> 种类适用 </td><td> 简要描述;简介 </td><td> 参考 </td></tr><tr><td>牛油</td><td>筛选/表征</td><td>欧洲的地理起源</td><td> 15 </td></tr><tr><td>酸奶</td><td>生物过程监控</td><td>乳酸过程中的进化心理状态</td><td> 16 </td></tr><tr><td>谷物酒吧</td><td> 体内测量</td><td>在食用不同糖成分的谷物酒的食物空间</td><td> 17 </td></tr><tr><td>液体模型系统</td><td>模拟口服条件</td><td>口腔压力和口腔状况的评估</td><td> 18 </td></tr><tr><td>苹果</td><td> 体内测量</td><td>消费苹果中的空白具有不同的遗传，结构和物理化学参数</td><td> 19 </td></tr><tr><td>咖啡</td><td>筛选/表征</td><td>专业咖啡的区别</td><td> 20 </td></tr><tr><td>葡萄必须</td><td>筛选/表征</td><td>烹饪过程的影响</td><td> 21 </td></tr><tr><td>调味糖果</td><td> 体内测量</td><td>决定使用不同的小组成员直接质谱法</td><td> 22 </td></tr><tr><td>火腿</td><td>筛选/表征</td><td>养猪系统的效果</td><td> 23 </td></tr><tr><td>面包</td><td>模拟口服条件</td><td>在咀嚼期间模拟面包香气</td><td> 24 </td></tr><tr><td>牛奶</td><td>筛选/表征</td><td>监测牛奶中光氧化诱导的动态变化</td><td> 25 </td></tr><tr><td>咖啡</td><td>筛选/表征</td><td>来自不同地理起源的烤咖啡的多样性</td><td> 26 </td></tr><tr><td>面包</td><td>生物过程监控</td><td>不同酵母起泡器在酒精发酵过程中的作用</td><td> 27 </td></tr><tr><td>咖啡</td><td> 体内测量</td><td>在消费不同的焙炒咖啡制品期间的空气</td><td> 28 </td></tr><tr><tD&gt;西红柿</td><td>筛选/表征</td><td>生产位置，生产系统和品种的影响</td><td> 29 </td></tr><tr><td>面包</td><td>生物过程监控</td><td>酒精发酵过程中面粉，酵母及其相互作用的影响</td><td>三十</td></tr><tr><td>蘑菇</td><td>筛选/表征</td><td>干燥的猪笼草蘑菇的保质期</td><td> 31 </td></tr><tr><td>酸奶</td><td>生物过程监控</td><td>不同发酵剂培养物在乳酸发酵过程中的作用</td><td> 32 </td></tr><tr><td>苹果</td><td>筛选/表征</td><td>苹果种质资源多样性</td><td> 33 </td></tr><tr><td>咖啡</td><td>筛选/表征</td><td>跟踪咖啡来源</td><td> 34 </td></tr><tr><td>咖啡</td><td> 体内测量</td><td>组合a动态感官方法和体内鼻子空间分析来了解咖啡感觉</td><td> 35 </td></tr></tbody></table> 表1:使用PTR-ToF-MS在食品行业的科学研究清单。使用基于PTR的方法在食品相关实验中监测VOC含量的科学研究非详尽列表。 在最近的研究中，我们报告了P​​TR-ToF-MS的应用以及自动采样系统和定制的数据分析工具，以提高采样自动化和可靠性，从而提高这种技术的潜力7,10,13。这允许我们在VOC含量方面筛选大样本集（ 例如具有许多重复的不同来源的食物，整个胚泡），以分析几种实验模式对VOC释放的影响（ 例如不同浓度特定技术参数的不同强度），并监测与给定生物过程相关的VOC（ 如酶氧化，酒精发酵）。在这里，为了说明PTR-ToF-MS在农业食品行业的潜力，我们提出了三种范式应用:检测由不同微生物启动子培养物诱导的酸奶乳酸发酵期间释放的VOC（在线生物过程监测），监测与不同苹果栽培品种（大规模筛选）相关的挥发性有机化合物（VOC），以及饮用咖啡时鼻内VOC释放的体内研究（鼻孔空间分析）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PTR-TOF 8000 High-Resolution PTR-TOF-MS</td>
			<td>Ionicon Analytik Ges.m.b.H.</td>
			<td>PTR-TOF 8000</td>
			<td>An detector for volatile organic compounds (VOCs) that allows for continuous VOC quantification with a very high mass resolution</td>
		</tr>
		<tr>
			<td>GERSTEL&#160;MPS 2XL</td>
			<td>Gerstel</td>
			<td></td>
			<td>A multifunctional autosampler&#160;</td>
		</tr>
		<tr>
			<td>Gas Calibration Unit</td>
			<td>Ionicon Analytik Ges.m.b.H.</td>
			<td>GCU-s / GCU-a</td>
			<td>A dynamic gas dilution system that provides variable but known quantities of different standard compounds in a carrier gas stream</td>
		</tr>
		<tr>
			<td>TofDaq</td>
			<td>Tofwerk AG</td>
			<td>free available at http://soft.tofwerk.com/&#160;&#160;&#160;</td>
			<td>A data acquisition software&#160;(for spectra&#160; acquisition)</td>
		</tr>
		<tr>
			<td>MATLAB&#160;</td>
			<td>MathWorks</td>
			<td>http://it.mathworks.com/products/matlab/</td>
			<td>A technical computing language and interactive environment for algorithm development, data visualization, and data analysis</td>
		</tr>
		<tr>
			<td>R</td>
			<td>The R Foundation</td>
			<td>free available at https://cran.r-project.org/mirrors.html&#160;&#160;</td>
			<td>A language and environment for statistical computing and graphics</td>
		</tr>
	</tbody>
</table>

ptr-tof-ms coupled with an automated sampling system and tailored data analysis for food studies: bioprocess monitoring, screening and nose-space analysis

质子转移反应（PTR），结合飞行时间（ToF）质谱（MS）是基于直接注入质谱（DIMS）技术的基于化学电离的分析方法。这些技术可以快速测定挥发性有机化合物（VOC），确保高灵敏度和准确度。一般来说，PTR-MS既不需要样品制备也不需要样品破坏，可以对样品进行实时和非侵入性分析。 PTR-MS在许多领域得到开发，从环境和大气化学到医学和生物科学。最近，我们开发了一种基于PTR-ToF-MS联合自动采样器和定制数据分析工具的方法，以增加自动化程度，从而提高该技术的潜力。这种方法允许我们监测生物过程（ 例如酶促氧化，酒精发酵），筛选大样品组例如不同的来源，整个胚泡），并分析几种实验模式（ 例如 ，给定成分的不同浓度，特定技术参数的不同强度）在VOC含量方面。在这里，我们报告了实验方案，例示了我们的方法的不同可能的应用: 即酸奶发酵过程中释放的VOCs（在线生物过程监测），与不同苹果品种（大规模筛选）相关的VOC的监测， ，以及咖啡饮用期间维生素VOC释放的体内研究（鼻孔空间分析）。

<html>样品的挥发性曲线导致了每秒获得的所需质量范围的完整质谱。在图2中 ，给出了酸奶在线生物过程中获得的平均光谱的一个例子32 。在每个频谱中，可以识别在m / z范围内高达250 Th的300个质量峰。 <img alt="图2" src="/files/ftp_upload/54075/54075fig2.jpg" /> 图2: 酸奶制造过程中接?...

Watch this Scientific Journal Video about PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis at 

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

PTR-ToF-MS结合自动采样系统和食品研究定制数据分析:生物过程监测，筛选和鼻空间分析

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

ptr-tof-ms-coupled-with-an-automated-sampling-system-tailored-data

Research

JoVE Journal

Chemistry

12.3K 次观看.  Fondazione Edmund Mach (FEM). Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

视频: PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

科研

化学

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

LC-MCYMS 诊断碎裂过滤的天然产物发现: 微囊藻毒素分析的应用

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

通过实验室间比较，对液态介质中的金纳米粒子进行纳米粒子跟踪分析

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

A number of different procedures for preparing nanoparticles for surface analysis are presented (drop casting, spin coating, deposition from powders, and cryofixation). We discuss the challenges, opportunities, and possible applications of each method, particularly regarding the changes in the surface properties caused by the different preparation methods.

用于ToF-SIMS和XPS分析的纳米颗粒的制备

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, ...