Name: tuning a parallel segmented flow column and enabling multiplexed detection
Uploaded: 2015-12-15T15:00:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection at JoVE.com

This work was supported by UWS and ThermoFisher Scientific. One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

注:该协议包含有关如何使用PSF列上加上多个探测器多重检测高效液相色谱系统的说明。该协议已被写入假设读者已经在色谱仪及各种HPLC检测方法的基本知识和经验。 注意:请参阅材料安全数据表（MSDS）的所有材料和试剂使用前（ 即 MSDS甲醇）。确保使用一切适当的安全措施处理溶剂和高效液相色谱法（HPLC）洗脱时。确保适当使用高效液相色谱仪，分析天平和检测仪器仪表工程控制，保证使用个人防护用品（防护眼镜，手套，实验室外套，全长长裤和封闭趾鞋）。 1.设置高效液相色谱仪<ol><li>准备高效液相色谱仪，用超纯水（例如，100％的Milli-Q水）林Ea和100％甲醇的线B作为流动相和清除泵按照制造商的要求。如果所使用的检测器之一是MS的，因为它是在这里，添加0.1％甲酸两者流动相A和B的</li><li>设置HPLC仪器组件和检测器，如图2。这需要相对于所述柱的探测器方便放置以便最小化检测器和塔之间的死体积。灵活性在HPLC系统配置是可取的。 </li></ol> 2.设置的UV-Vis和MS检测器<ol><li>设定UV-Vis检测到所需的波长依赖于感兴趣的样品（例如，280纳米）。 </li><li>坐落在积极模式总离子计数（TIC）分析，采用全扫描检测方法的质谱检测器。还可以调整以下的MS参数，因此:蒸发器温度500℃，毛细管温度350℃，鞘气设定的速度为60台，辅助气流40扫了5台气流和喷雾电压3.5千伏。这些设置可以在以后的特定的用户要求依赖于被分析的样本进行调节。 </li></ol> 3.制备2,2-二苯基- 1 -苦味自由基（DPPH•）试剂DPPH•系统检测器的设置<ol><li>权衡的DPPH•25毫克，溶解在250ml甲醇中在容量瓶中。 </li><li>加入250微升甲酸的DPPH•试剂。盖上烧瓶中箔，以防止暴露在光线下。 </li><li>超声处理含有DPPH•试剂进行10分钟的烧瓶中。 </li><li>清除DPPH•泵的准备DPPH•试剂根据制造商的要求。 </li><li> 根据图2通过将泵线到一个T形件的入口设置的DPPH•系统。 </li><li>附加100微升反应管为thT型件电子出口并附加反应线圈到检测器的另一端。 </li><li>包住反应线圈在柱加热器和设置列加热器温度到60℃。 </li><li>设置DPPH•UV-Vis检测到520纳米。 </li></ol> 4.设置PSF柱<ol><li>涤纶短纤柱的入口连接到高效液相色谱仪。 </li><li>中央端口连接到质谱仪，使用0.13毫米内径的管道有15厘米长。 </li><li>一个外围端口连接到使用的0.13毫米ID管15厘米长的UV-Vis检测。 </li><li>连接其他外设端口连接到T型件使用0.13毫米内径的管道有15厘米长的DPPH•检测系统。 </li><li>使用柱塞阻止未使用的外设端口。 </li><li>带来的HPLC泵的流速到1ml分钟-1，在100％的线B - 100％甲醇（0.1％甲酸）。 </li><li>平衡与100％甲醇的移动ph值列ASE 20分钟为4.6毫米内径×250毫米长度列。根据其他列的用户可以采用的尺寸此时间缩放。 </li></ol> 5.调优PSF列的多重检测<ol><li>测量的至少两个空收集容器（一个用于连接到UV-Vis检测器，一个用于DPPH•检测器的端口）使用分析天平的质量。 </li><li>收集紫外可见的DPPH•端口流动相为两个单独的，预先称重的收集容器（5.1）。记录的时间段为集合。收集至少500毫克的溶剂中，在每一个容器中。 </li><li>称量收集容器并确定流动相的质量。定甲醇的密度0.791克毫升-1，确定从每个端口收集流动相的体积。 </li><li>通过差，即，标称泵设定流率减去通过DPPH•流速和紫外可见DETECTOř流动口，确定流率到MS检测器。表示每个流动比例为总流量的百分比。 注意:在理想情况下，则流程百分比均为:向MS是总流量的18％，到紫外可见22％，到DPPH•探测器60％。 </li><li>如果不是这样，通过改变紫外 - 可见检测器上的背压调节流量的百分比。例如，如果流至紫外可见太高，通过添加加0.13毫米ID管15厘米部分到UV-Vis检测器的出口降低的比例。然后重复步骤5.1至5.5。 </li></ol> 6.最终设置条件<ol><li>设置DPPH•试剂泵到相同的流速流出连接到DPPH•检测器的出口的流速。 注:复用紫外-可见分光光度计的PSF列，DPPH•和MS现在可以进行分析。 </li></ol>

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The authors have nothing to disclose.

这项研究涉及的表征和使用高效液相色谱多重检测采用平行分段流动（PSF）列咖啡剖析。复使用的HPLC柱的PSF使关键化学实体的表征和鉴定通过减少而它需要使用常规的多检测处理的一小部分时间内获得更大程度的分子的特定信息的样品的数据的复杂性。该PSF列不仅可为多重检测平台，同时还兼具破坏性和非破坏性检测相结​​合，无需额外的死体积和管道。 DPPH•，UV-Vis和MS（TIC）进行多...

主动流技术专栏 主动流技术（AFT）层析柱最近开发来克服低效率与流量异质1-6相关联的分离并且还使多重检测。在这个特殊的沟通我们详细介绍了并行的操作过程分段流程（PSF）柱多重检测。 PSF中列的关键的功能的优点是:（1）从柱床的径向中心区域的流动被从流动的周边或壁区域分离，（2）流动相必须由一个检测要处理的体积源被还原，和（3）检测源可以复用，以未经灌输检测延迟在每个检测处理，或随后迫使一个柱后流动流7,8的分束扩大样品信息。 PSF中柱设计中的关键功能，使一个多重检测的dvantage是小说出口配件和玻璃料组件，图1是在船尾柱的照片比现有柱。重要的是要明白，分裂过​​程使用并行分段流动列是不一样的柱后流流拆分获得是重要的。在后柱流动流分割整个样本频带从前缘到尾的均等取样的四肢（即，轴向地），从而，每一个流动流等于相对于效率和灵敏度;灵敏度的幅度由此被除以分割数。在PSF，然而，分割处理的样品带径向，轴向未。因此，中央口采样峰顶 - 高峰最集中的地区。因此，这里的灵敏度最高的峰不被漫拖尾区域稀释。将样品从外围端口洗脱是效率不如在CENTRA升区，但是，因为带被径向采样，而不是沿轴向，所述峰的宽度窄于将一个采样过程，其将上述峰在轴向上，也就是 ，一柱后分裂的情况。因此与浓度相关的检测器的灵敏度不会降低。 在PSF列，出口配件包括多个出口端口，并在此端部接头内部有坐落在一个环形的玻璃料。的该环形熔料通道的内部流出通过所述径向中心出口端口之列，而出口料通道的外径向部分经由外围或壁区域出口流动口流出的列。出口玻璃料的内，外部分由不可渗透的阻挡层，防止这些流区 2之间横向流动分离。作为该设计的结果通过该柱床的中央径向流动物流从壁面区域流动插件分开IDE列。流来自这两个区域的相对部分可通过压力管理，以便优化所述柱技术的各种功能方面，例如分离效率或检测灵敏度变化以几乎任何期望的比例。从本质上说，这种设计有效地较大的格式列一个"虚拟"的列内建立，具有较窄内径，因此，列函数作为一个真正的无壁塔，克服柱床异质和壁效应9,10。 PSF列的主要好处是提高柱效，尽量减少溶剂处理的检测源（S），使多路检测。然而，一个额外的优点是，由于任何带的拖尾和面向地区被从整体洗脱除去轮廓溶质在洗脱或检测存在于更高浓度比原本为相同的S观察到的olute注射和浓度负载在常规柱，根据所使用的分割比。其结果往往存在观察到的信号强度的增益对PSF列2进行分离。事实上，如果分割比率进行调整，使得从每四个出口的流出口的25％，即使用紫外线（UV）检测中观察到的信号强度几乎示出使用常规的完全相同的信号强度作为表观其中移动相的整个（100％），分析7列 。此外，中央和壁流区域之间的出口比微调允许优化该列的效率。 （1）的流量，（2）的分割比率，以及（3）溶质保留因子:在柱效增益使用AFT列不能被说成一个单一的值，因为这些效率增益是三个因素的函数观察。然而，提高效率相比，CONVentional列几乎总是观察到，有时这些收益是在理论塔板1,2的数量超过了100％。的能力来调整分割比允许分析者有效地定制"虚拟"塔的直径，这是相对于检测处理的一个重要因素。例如，一个虚拟2.1毫米内径（ID）列是从一个物理4.6毫米内径柱成立时的分割比是流动相从径向中央出口洗脱的21％。在这些条件下，虚拟2.1毫米内径柱执行与效率可能比以往的2.1毫米内径柱大于70％以上，这取决于流速，和溶质保留因子10。 一个用于多路检测的当前的PSF柱设计集成了一个4端口出口配件，但该列可以配备2端口端配合也，然而，这限制了检测吨Ø只有两个探测器。这些列的基本操作是，然而，同样的，所不同的是四个探测器可以同时耦合到4端口出口的PSF列范围扩大为多重检测。除了前和后柱连接管，唯一的附加的要求进行操作PSF中列管可连接到外围出口端口，并通过该通过各管流动相的量可​​以被测量的装置，通常是一个质量测量或体积测量。为了便于调谐，所有出口流管的内径应该是相同的。圆周和径向中央出口端口之间的流量比，然后通过使用压力管理的变化，简单地通过改变位于所述外围出口配件，或管道交检测器上的径向中央出口孔的长度的管道的长度。 多重检测应用PSF列 </stroNG&gt; PSF列的一个重要优点是，每一个的出口退出端口可直接连接到检测源，从而使多路检测。在设计良好的检测系统具有多路检测的单个分析可以提供在问候样本内的组分的性质基本信息。重要的是，破坏性和非破坏性测试可以精确地在同一时间进行，而不检测延迟。这允许的绝对分配，例如使用DPPH•试剂，与组件观察以洗脱用UV和/或质谱（MS）检测响应7,11抗氧化剂。因此，四个独立检测器可同时与水流通过任何四个出口端口引导到每个检测器适当部分进行操作。因为通过这些端口的流动可以容易地调节溶质到达任意检测器中的量可被调节以适合给定的检测器源的灵敏度。应当指出，然而，最有效的溶质迁移通过径向中央出口端口观察。每个外围设备端口提供等同的分离效率，其中当通过每个端口设置为25％，仅比传统柱略有效率较低。因此，重要的是定量检测器被设置为从径向中央出口分析样品。 当设置用于多路复用检测目的一个PSF列有一些需要被制成以实现高效率和高品质的效果方面的考虑;选择哪个端口的一种类型的检测器和流量调节的是管的尺寸为每个端口，。 管外形尺寸为每个端口 在层析柱后的管道长度起着分离的效率和性能至关重要的作用。大DEA的d体积从柱出口到检测器长或宽的id管的结果，将导致效率，分辨率和灵敏度的损失。因此，设置在PSF柱以达到最大的潜力在提供有效的分离，同时提供多路复用的好处时适当管道尺寸必须被利用。 港探测器 图2是多重检测（紫外-可见（U -可见光），质谱（MS）和2,2-二苯基-1-苦基 ​​苯肼（DPPH•）检测）的一个例子的设置的图示。该图显示了中央端口附连到质谱仪，而DPPH•和紫外-可见检测器被附连到外设端口。由于MS是三种最敏感的检测器，流动到该检测器从中央出口端口被指挥。由于DPPH•检测是选择性的presencË的抗氧化剂，和最不敏感和最耐受谱带增宽，流到这个检测器从一个外围端口被指挥。紫外 - 可见是次要的"通用"探测器，因此流向该探测器从第二外设端口的导演。 流量调节 一旦适当的管道已被附接到从一个端口到检测器，从各检测器的排出流量可调节到所需的量。测量流量从每个检测器排出的量的简单方法是称量流动相的洗脱通过每个端口上的给定时间段的量。的百分比流量因此可确定，并且通流比可通过缩短或延长附着于检测器的出口管线管据此以适合所选择的检测器的要求进行调整。不同的检测器具有流量不同的要求，例如，流动细胞荧光检测器（FLD）不流速的限制，但必须小心以避免过度加压流通池。因此流过FLD的控制通常是通过调整穿过其他检测器的压降和流量的剩余部分然后通过FLD传递实现。的检测器即流被递送的量敏感是MS。一般情况下，目前的高端质谱仪可以很容易地处理大约1〜1.5毫升/中度水流动相分。上述该流速，源的驱可能使该MS无法操作。然而，在大多数质谱仪检测灵敏度受益通过使用较低的流速;因此，PSF分流能力是涉及MS检测应用中非常有用。高柱体积流速可以利用，但随着运送到质谱仪低容量负载。流动到MS检测器的调谐，然而，必须通过调整前向压降制成质谱检测器，而不是后期的MS。这里，使用窄孔管（0.1毫米内径）是非常有用的，因为压力可以在不增加不适当的死体积容易地调节。 取决于检测器的类型的分割比的调整既可以做前或后检测器。如果一个非破坏性的检测器，如紫外可见时，流量百分比将被测量和调整交检测器。如果单个检测器破坏性被用在多重设置的流百分比通过回计算相对于其他端口流动百分比来确定。如果一个试剂基于检测器的使用，例如DPPH•，流程百分比被测量不加入试剂后检测器;如果两个或更多的破坏性检测器被使用，则流量比被测量预检测器。检测系统可能需要额外的仪器仪表等DPPH•，将有额外的系统压力可能改变流动百分比一旦附着到检测系统。因此，慎重考虑应支付给破坏性检测系统压力，调节流量百分比时，预先检测。不论是通过任何端口设定的流量比的，定量信息应通过适当的标准化而获得。一旦流量比设置，但是，它们是健壮的，他们甚至不下的梯度洗脱条件7改变， 详细的视频协议附带的这篇稿件的目的是展示如何在检测的复用方式，使用和调整PSF列运作。

<table><tbody><tr><td>HPLC instrument</td><td></td><td></td><td>Multiple detectors of choice for multiplexed detection. Detectors of choice may require additional instrumentation, &lt;em&gt;e.g.&lt;/em&gt;, pump.</td></tr><tr><td>Parallel Segmented Flow HPLC column</td><td>Thermo Fisher Scientific</td><td>Not Defined</td><td>Soon to be commercialized</td></tr><tr><td>Methanol</td><td>Any brand</td><td></td><td>HPLC Grade</td></tr><tr><td>PEEK tubing</td><td>Any brand</td><td></td><td>Various lengths and i.d.</td></tr><tr><td>Column stoppers</td><td>Any brand</td><td></td><td>For blocking unused peripheral ports.</td></tr><tr><td>PEEK tube cutter</td><td>Any brand</td><td></td><td></td></tr><tr><td>Analytical Scale Balance</td><td>Any brand</td><td></td><td></td></tr><tr><td>Stop watch</td><td>Any brand</td><td></td><td></td></tr><tr><td>Eluent collection vessels</td><td>Any brand</td><td></td><td>1-2 ml sample vials can be used as eluent collection vessels</td></tr></tbody></table>

tuning a parallel segmented flow column and enabling multiplexed detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

一个复用HPLC分析进行了使用PSF模式（ 图1）的后列，并设置为如图 2所示。这种类型的设置允许的咖啡样品同时通过UV-Vis，DPPH•和MS的总分析离子计数（TIC）模式。咖啡样品作出答复DPPH•的化合物然后可以很容易地匹配到紫外可见和MS -基于保留时间对准的TIC响应，如图3中，由于同时记录色谱图。其中正响应来自MS-TIC检测器看到?...

Watch this Scientific Journal Video about Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection at JoVE.com

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

调优并行分段流动列和启用多重检测

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms ...

tuning-parallel-segmented-flow-column-enabling-multiplexed

Research

JoVE Journal

Chemistry

7.3K 次观看.  University of Western Sydney. Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection. 

视频: Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

柱后衍生用反应流高效液相色谱柱

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

化学

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

幕式柱:效率和灵敏度的优化

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

微流控成像流式细胞仪通过不对称检测时间延伸光学显微镜（ATOM）

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

工程学

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

胶体半导体纳米晶系统研究的模块化微流控技术

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.