Name: paper-based preconcentration and isolation of microvesicles and exosomes
Uploaded: 2020-04-29T15:00:00
Description: Watch this Scientific Journal Video about Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes at JoVE.com

这项研究得到了韩国国家研究基金会资助，授予NRF-2018R1D1A1A09084044。2019年，J.H.Lee得到了光格永大学的研究资助。金海林得到了韩国贸易、工业和能源部（MOTIE）的"工业专家能力发展计划"的支持，该方案由韩国技术促进研究所（KIAT）运营。P0002397，可穿戴智能设备工业融合人力资源开发计划）。

1. 设备制造
<ol><li>使用打印机软件（材料表）定义要在纸张上打印区域。 注：该设计有12个蜡纹层，其中圆形样品区域的直径逐渐从5毫米缩小到2毫米（图1A）。</li>
	<li>使用商业蜡印机（材料表）在纤维素纸（材料表）两侧的指定区域上打印疏水蜡（图1B）。</li>
	<li>将蜡印纸在 120 °C 下放入实验室烤箱中 80 度...

<ol>
	<li>Edgar, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q+&amp;+A:+What+are+exosomes,+exactly.">Q &amp; A: What are exosomes, exactly.</a> BMC Biology. 14 (1), 1-7 (2016).</li><li>Contreras-Naranjo, J. C., Wu, H. J., Ugaz, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+exosome+isolation+and+analysis:+Enabling+liquid+biopsy+for+personalized+medicine.">Microfluidics for exosome isolation and analysis: Enabling liquid biopsy for personalized medicine.</a> Lab on a Chip. 17 (21), 3558-3577 (2017).</li><li>Simons, M., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+-+vesicular+carriers+for+intercellular+communication.">Exosomes - vesicular carriers for intercellular communication.</a> Current Opinion in Cell Biology. 21 (4), 575-581 (2009).</li><li>St&#229;hl, A. L., Johansson, K., Mossberg, M., Kahn, R., Karpman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+microvesicles+in+normal+physiology,+pathophysiology,+and+renal+diseases.">Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases.</a> Pediatric Nephrology. 34 (1), 11-30 (2019).</li><li>Chen, C., Lin, B. R., Hsu, M. Y., Cheng, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+devices+for+isolation+and+characterization+of+extracellular+vesicles.">Paper-based devices for isolation and characterization of extracellular vesicles.</a> Journal of Visualized Experiments. (98), e52722 (2015).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origami-paper-based+device+for+microvesicle/exosome+preconcentration+and+isolation.">Origami-paper-based device for microvesicle/exosome preconcentration and isolation.</a> Lab on a Chip. 19 (23), 3917-3921 (2019).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+Exosomes,+microvesicles,+and+friends.">Extracellular vesicles: Exosomes, microvesicles, and friends.</a> Journal of Cell Biology. 200 (4), 373-383 (2013).</li><li>Lee, Y., El Andaloussi, S., Wood, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+microvesicles:+Extracellular+vesicles+for+genetic+information+transfer+and+gene+therapy.">Exosomes and microvesicles: Extracellular vesicles for genetic information transfer and gene therapy.</a> Human Molecular Genetics. 21, 125-134 (2012).</li><li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-Free+Isolation+of+Exosomes+from+Extracellular+Vesicles+by+Microfluidic+Viscoelastic+Flows.">Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows.</a> ACS Nano. 11 (7), 6968-6976 (2017).</li><li>Marczak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+isolation+and+preconcentration+of+exosomes+by+ion+concentration+polarization.">Simultaneous isolation and preconcentration of exosomes by ion concentration polarization.</a> Electrophoresis. 39 (15), 2029-2038 (2018).</li><li>Livshts, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+exosomes+by+differential+centrifugation:+Theoretical+analysis+of+a+commonly+used+protocol.">Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.</a> Scientific Reports. 5, 1-14 (2015).</li><li>Chiriac&#242;, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-chip+for+exosomes+and+microvesicles+detection+and+characterization.">Lab-on-chip for exosomes and microvesicles detection and characterization.</a> Sensors. 18 (10), 3175 (2018).</li><li>Lobb, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> Journal of Extracellular Vesicles. 4 (1), 1-11 (2015).</li><li>Taylor, D. D., Shah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolating+extracellular+vesicles+impact+down-stream+analyses+of+their+cargoes.">Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.</a> Methods. 87, 3-10 (2015).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetic+size-based+spatial+separation+of+micro/nanospheres+using+paper-based+3d+origami+preconcentrator.">Electrokinetic size-based spatial separation of micro/nanospheres using paper-based 3d origami preconcentrator.</a> Analytical Chemistry. 91 (16), 10744-10749 (2019).</li><li>Yeh, S. H., Chou, K. H., Yang, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+pre-concentration+with+high+enrichment+factors+at+a+fixed+location+in+paper-based+microfluidic+devices.">Sample pre-concentration with high enrichment factors at a fixed location in paper-based microfluidic devices.</a> Lab on a Chip. 16 (5), 925-931 (2016).</li></ol>

虽然 Exo-PAD 已成功用于显微和外显子的富集和分离，但应仔细考虑几个关键点：1） 设备制备期间的烤箱孵化时间和温度，2） 处理时间，3） 施加不同层数和样品面积直径的电压，以及 4） 适用于临床样品。
协议给出的孵化时间和温度是制造可靠器件的优化条件。由于印刷蜡的回流过多，较长的孵育时间或较高的温度可能会导致收敛设计失真。这将防止聚焦电场线路的形成?...

微囊和外显体是小膜囊泡，分别测量0.2±1μm和30±200nm。它们被,几个不同的细胞类型,1、2、3、4、53,分分泌入细胞,外环境。2它们含有DNA、mRNA、miRNA、蛋白质和脂质子集形式的亲细胞信息，通过各种体液（如血清、血浆、尿液、脑脊液、羊水以及唾液,,6、7、8、9）7在体内循环。68因此，从生物液体中有效分离微囊和外体的技术可以在疾病的诊断、预后和实时监测以及开发新疗法方面提供广泛的机会。
然而，传统的基于超离心的微囊和外体分离方法非常耗时，并导致样品的显著损失和污染。这是因为它涉及几个繁琐的移液和装载步骤和丢弃各种试剂与重复的超离心5，6，10，11，12。6,10,11,125此外，超离心引起的高剪切应力（±100，000 x g）可导致微囊和外显子的物理解损伤，产生不良的回收率（5~23%）6、13、14。6,13,14因此，必须开发一种高效、不显眼的微囊和外体隔离技术，以减少损伤和损失，从而实现更高的回收率。
一种以折纸为基础的装置（Exo-PAD）被开发用于简单、温和、高效分离微囊和外显体6。Exo-PAD 的设计是一种多面纸，具有连续连接的样品区域，直径逐渐减小。离子浓度极化（ICP）技术是一种纳米电动力学现象，预浓缩带电生物分子，并融入这一独特的设计。使用 Exo-PAD 可使特定层中的微囊和外体富集五倍，并且只需展开设备即可进行隔离。本文详细介绍了Exo-PAD，从整体器件的制造和操作到分析其使用，以说明该方法并展示具有代表性的结果6。

<table border="0" cellpadding="0" cellspacing="0" style="width:1356px;" width="1355">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:397px;">Ag/AgCl electrodes</td>
			<td style="width:207px;">A-M Systems, Inc.</td>
			<td style="width:119px;">531500</td>
			<td style="width:633px;">0.15&#34; diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumin from Bovine Serum (BSA), Alexa Fluor 594 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A13101</td>
			<td>BSA conjugated with Alexa Fluor 594 (Ex/Em: 590/617 nm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbonate-Bicarbonate Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50CAP</td>
			<td>Carbonate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CorelDraw software (Coral Co., Canada)</td>
			<td>Corel Corporation</td>
			<td></td>
			<td>Printer software to define wax printing region</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ColorQube 8870</td>
			<td>Xerox Corporation</td>
			<td></td>
			<td>Wax printer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromatography paper grade 1</td>
			<td>Whatman</td>
			<td>3001-861</td>
			<td>Cellulose paper, dimension: 20 * 20 cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent-labeled exosome standards</td>
			<td>HansaBioMed Life Sciences, Ltd.</td>
			<td>HBM-F-PEP-100</td>
			<td>Exosome labeled with FITC (Ex/Em: 490/520 nm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Keithley 2410 current/voltage source-meter</td>
			<td>Keithley Instruments, Inc.</td>
			<td style="width:119px;"></td>
			<td>Current&#8211;voltage source measurement system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nafion perfluorinated resin solution</td>
			<td>Sigma-Aldrich</td>
			<td>31175-20-9</td>
			<td>Permselective membrane, 20 wt.% in the mixture of lower aliphatic alcohols and water; contains 34% water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoSight LM10</td>
			<td>NanoSight Technology</td>
			<td style="width:119px;"></td>
			<td>Nanoparticle tracking analysis (NTA) machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS, pH7.4)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010001</td>
			<td></td>
		</tr>
	</tbody>
</table>

paper-based preconcentration and isolation of microvesicles and exosomes

微囊和外体是小膜囊泡释放到细胞外环境，并在整个身体中循环。由于它们含有各种亲细胞衍生生物分子，如DNA、mRNA、miRNA、蛋白质和脂质，因此它们的浓缩和分离是它们作为临床应用的潜在生物标志物被开发的关键步骤。然而，传统的分离方法（例如，超离心）对微囊和外显体造成重大损失和损害。这些方法还需要多个重复步骤的超离心，加载和浪费试剂。本文介绍了一种详细的方法，以制造一个折纸为基础的设备（Exo-PAD），旨在有效地丰富和隔离微囊和外体在简单的方式。Exo-PAD 的独特设计由手风琴样多层和收敛样品区组成，与离子浓度极化技术集成，从而使特定层的微囊和外显体具有五倍的富集。此外，通过展开 Exo-PAD，分离出富集的微囊和外显子。

必须优化操作时间，以实现富集的微囊和外显体的最大回收率。时间不足不允许足够的微囊和外体迁移，从而减少富集，而过多的时间会恶化空间聚焦，从而分散微囊和外体。因此，通过时间优化步骤，可以确定微囊和外体的最大预集中系数以及微囊和外体最丰富的最终位置。为了找到微量和外显子的最终位置，用显微镜观察了荧光标记微量微量和外显子的延时迁移（图2A），</str...

Watch this Scientific Journal Video about Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes at JoVE.com

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

提供是一种制造纸基装置的协议，用于有效丰富和分离微囊和外显体。

微量和外显子的纸基预集中和分离

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

在此协议中，我们描述了一种简单的方法来制造一种称为XO Pad的纸基设备，该器件设计用于分离细胞外囊泡进行有效浓缩。XO Pad 是一种简单的设备，无需使用任何化学品或工作机器即可预浓缩和分离细胞外囊泡，同时显示比传统方法更高的回收率。首先定义要使用打印机软件打印的区域。设计应有12个蜡图案层，其中圆形样品区域的直径逐渐从5毫米缩小到2毫米。然后，在纤维素纸两侧的指定区域上打印疏水蜡。将蜡印纸在实验室烤箱中放置 80 秒，温度为 120 摄氏度，然后用切割机切割蜡印纸，以制作单独的 XO Pad 设备。将五升和两微升的渗透选择性膜分别滴到最左侧和最右边最层的样品区域。将用渗透选择性膜编码的层在70摄氏度的热板上放置30分钟，以蒸发溶剂。然后用压力敏感胶带密封涂层层最外层，用压力敏感胶带密封缓冲液，留下一个小孔。沿白线来回折叠 XO Pad 设备，以收敛连接所有采样区域。在收敛样品区域中移液 15 微升微升微量和外显体样品，等待几秒钟以确保完全润湿。将两个丙烯酸腔室放在 XO 垫的两端，用小粘结夹将其牢固地夹住以防止展开。在腔室中填充 110 微升 0.1X PBS，并插入两个银电极。然后使用电流电压源测量系统对电极施加 30 伏，20 分钟。要分离富集的微囊和外体，请将折叠的 XO Pad 从丙烯酸室中分离，并展开设备，将富集的颗粒与其他层分离。冲出第8层和第9层的样品区域，其中微囊和外显体被丰富用于下游分析。对荧光标记的微囊和外显子进行了延时迁移测定，以优化操作时间，最大限度地回收富集颗粒。所有样品区域的荧光强度都用 ImageJ 软件进行量化。在富集过程之前，微囊和外显子分散在所有样品区，强度逐渐降低。经过10分钟的处理，颗粒以电基方式迁移，第七层出现即时预浓缩插头。当时间达到20分钟时，微囊和外体强烈聚焦在8层和9层，并基于荧光强度丰富了5倍。用SEM对第8层富集微囊和外显体的完整性进行了调查，在富集过程后观察到更多的非损伤颗粒。还分析了富集微囊和外显子的大小分布。随后，对6层、8层和10层样品区富集微囊和外显体的实际浓度进行了分析。在第8层，微囊和外体预浓缩5.58，但在其他层中未观察到明显的预浓度。为确保本程序的可重复性，在设备验证和设备运行时间期间精确遵循烤箱孵育时间和温度协议，因为这些是优化的条件。XO Pad 还可用于使用碳酸盐缓冲液将细胞外囊泡从富含蛋白质的样品（如血清或血浆）分离和分离。该协议可以提供高效、简单的制备工具，具有临床研究所需的预集中和隔离功能，如诊断、药物输送、免疫治疗和其他治疗。

Research

JoVE Journal

Biochemistry

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

科研

生物化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Rapid Isolation of the Mitoribosome from HEK Cells

HEK 细胞 Mitoribosome 的快速分离

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Detection and Isolation of Apoptotic Bodies to High Purity

高纯度凋亡体的检测与分离

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

超中心对等离子脂蛋白的分离和分析

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...