This work was supported in part by the Taiwan National Science Council grants- NSC 99-2320-B-007-005-MY2 (CC) and NSC 101-2628-E-007-011-MY3 (CMC), and the Veterans General Hospitals and University System of Taiwan Joint Research Program (CC).

A general diagram of the operation procedure is provided in Figure 1. Using ethical practices, we collected blood samples from healthy subjects, and obtained aqueous humor samples from patients through the Taichung Veterans General Hospital (TCVGH), Taichung, Taiwan under IRB approved protocols (IRB TCVGH No. CF11213-1).
1. Fabrication of Paper Devices
<ol>
	<li>Cut chromatography paper into circles of 5 mm in diameter to provide the same layout as a 96-well microtiter plate. Sandwich these pape...

<ol>
	<li>Caby, M. P., Lankar, D., Vincendeau-Scherrer, C., Raposo, G., Bonnerot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal-like+vesicles+are+present+in+human+blood+plasma.">Exosomal-like vesicles are present in human blood plasma.</a> Int. Immunol. 17, 879-887 (2005).</li><li>Lasser, 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=Human+saliva,+plasma+and+breast+milk+exosomes+contain+RNA:+uptake+by+macrophages.">Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.</a> J. Transl. Med. 9, 9 (2011).</li><li>Raj, D. A. A., Fiume, I., Capasso, G., Pocsfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+quantitative+proteomics+strategy+for+protein+biomarker+studies+in+urinary+exosomes.">A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes.</a> Kidney Int. 81, 1263-1272 (2012).</li><li>Wiggins, R. C., Glatfelter, A., Kshirsagar, B., Brukman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procoagulant+activity+in+normal+human-urine+associated+with+subcellular+particles.">Procoagulant activity in normal human-urine associated with subcellular particles.</a> Kidney Int. 29, 591-597 (1986).</li><li>Admyre, 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=Exosomes+with+immune+modulatory+features+are+present+in+human+breast+milk.">Exosomes with immune modulatory features are present in human breast milk.</a> J. Immunol. 179, 1969-1978 (2007).</li><li>Keller, S., Ridinger, J., Rupp, A. K., Janssen, J. W. G., Altevogt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+fluid+derived+exosomes+as+a+novel+template+for+clinical+diagnostics.">Body fluid derived exosomes as a novel template for clinical diagnostics.</a> J. Transl. Med. 9, 86 (2011).</li><li>Asea, 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=Heat+shock+protein-containing+exosomes+in+mid-trimester+amniotic+fluids.">Heat shock protein-containing exosomes in mid-trimester amniotic fluids.</a> J. Reprod. Immunol. 79, 12-17 (2008).</li><li>Bard, M. P., 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=Proteomic+analysis+of+exosomes+isolated+from+human+malignant+pleural+effusions.">Proteomic analysis of exosomes isolated from human malignant pleural effusions.</a> Am. J. Resp. Cell Mol. Biol. 31, 114-121 (2004).</li><li>Perkumas, K. M., Hoffman, E. A., McKay, B. S., Allingham, R. R., Stamer, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocilin-associated+exosomes+in+human+ocular+samples.">Myocilin-associated exosomes in human ocular samples.</a> Exp. Eye Res. 84, 209-212 (2007).</li><li>Anderson, H. C., Mulhall, D., Garimella, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+membrane+vesicles+in+the+pathogenesis+of+various+diseases,+including+cancer,+renal+diseases,+atherosclerosis,+and+arthritis.">Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis.</a> Lab. Invest. 90, 1549-1557 (2010).</li><li>Montecalvo, 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=Mechanism+of+transfer+of+functional+microRNAs+between+mouse+dendritic+cells+via+exosomes.">Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.</a> Blood. 119, 756-766 (2012).</li><li>Grange, 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=Microvesicles+released+from+human+renal+cancer+stem+cells+stimulate+angiogenesis+and+formation+of+lung+premetastatic+niche.">Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche.</a> Cancer Res. 71, 5346-5356 (2011).</li><li>Jaiswal, R., 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=Microparticle-associated+nucleic+acids+mediate+trait+dominance+in+cancer.">Microparticle-associated nucleic acids mediate trait dominance in cancer.</a> Faseb. J. 26, 420-429 (2012).</li><li>Thery, C., Clayton, A., Amigorena, S., Raposo, G., Morgan, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in cell biology. (UNIT 3.22), (2006).</li><li>Rood, I. M., 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=Comparison+of+three+methods+for+isolation+of+urinary+microvesicles+to+identify+biomarkers+of+nephrotic+syndrome.">Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.</a> Kidney Int. 78, 810-816 (2010).</li><li>Taylor, D. D., Gercel-Taylor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+signatures+of+tumor-derived+exosomes+as+diagnostic+biomarkers+of+ovarian+cancer.">MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.</a> Gynecol. Oncol. 110, 13-21 (2008).</li><li>Witwer, K. W., 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=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> J. Extracellular Vesicles. 2, 20360 (2013).</li><li>Fernandez-Llama, P., 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=Tamm-Horsfall+protein+and+urinary+exosome+isolation.">Tamm-Horsfall protein and urinary exosome isolation.</a> Kidney Int. 77, 736-742 (2010).</li><li>Alvarez, M. L., Khosroheidari, M., Ravi, R. K., DiStefano, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein,+microRNA,+and+mRNA+yields+using+different+methods+of+urinary+exosome+isolation+for+the+discovery+of+kidney+disease+biomarkers.">Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.</a> Kidney Int. 82, 1024-1032 (2012).</li><li>Chen, 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=Microfluidic+isolation+and+transcriptome+analysis+of+serum+microvesicles.">Microfluidic isolation and transcriptome analysis of serum microvesicles.</a> Lab. Chip. 10, 505-511 (2010).</li><li>Davies, R. T., 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=Microfluidic+filtration+system+to+isolate+extracellular+vesicles+from+blood.">Microfluidic filtration system to isolate extracellular vesicles from blood.</a> Lab. Chip. 12, 5202-5210 (2012).</li><li>Chen, 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=Paper-based+immunoaffinity+devices+for+accessible+isolation+and+characterization+of+extracellular+vesicles.">Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles.</a> Microfluid. Nanofluid. 16, 849-856 (2014).</li><li>Lamparski, H. G., 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=Production+and+characterization+of+clinical+grade+exosomes+derived+from+dendritic+cells.">Production and characterization of clinical grade exosomes derived from dendritic cells.</a> J. Immunol. Methods. 270, 211-226 (2002).</li><li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+exosomes+and+HIV-1:+Purification+of+both+vesicles+from+cell-free+supernatants.">Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants.</a> J. Immunol. Methods. 338, 21-30 (2008).</li><li>Carrilho, E., Martinez, A. W., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+wax+printing:+a+simple+micropatterning+process+for+paper-based+microfluidics.">Understanding wax printing: a simple micropatterning process for paper-based microfluidics.</a> Anal. Chem. 81, 7091-7095 (2009).</li><li>Dungchai, W., Chailapakul, O., Henry, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+detection+for+paper-based+microfluidics.">Electrochemical detection for paper-based microfluidics.</a> Anal. Chem. 81, 5821-5826 (2009).</li><li>Glavan, A. 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=Omniphobic+'R-F+paper'+produced+by+silanization+of+paper+with+fluoroalkyltrichlorosilanes.">Omniphobic 'R-F paper' produced by silanization of paper with fluoroalkyltrichlorosilanes.</a> Adv. Funct. Mater. 24, 60-70 (2014).</li><li>Usami, S., Chen, H. H., Zhao, Y. H., Chien, S., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+construction+of+a+linear+shear-stress+flow+chamber.">Design and construction of a linear shear-stress flow chamber.</a> Ann. Biomed. Eng. 21, 77-83 (1993).</li><li>Murthy, S. K., Sin, A., Tompkins, R. G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+flow+and+surface+conditions+on+human+lymphocyte+isolation+using+microfluidic+chambers.">Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers.</a> Langmuir. 20, 11649-11655 (2004).</li><li>Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A., Ligler, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+chemical+cleaning+methods+of+glass+in+preparation+for+silanization.">Comparison of chemical cleaning methods of glass in preparation for silanization.</a> Biosens. 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The authors declare that they have no competing financial interests.

The most critical steps for successful isolation of subgroups of extracellular vesicles are: 1) a good choice of paper; 2) stable and high coverage of capture molecules on the surface of the paper fibers; 3) proper handling of samples; and 4) general laboratory hygiene practice.
Porous materials have been utilized in many inexpensive and equipment-free assays. They may have tunable pore size, versatile functionality, low cost and high surface-to-volume ratio permitting passive wicking of fluid...

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.
EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met. 
We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chromatography Paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3001-861</td>
			<td>&#160;Whatman&#174; Grade 1 cellulose paper</td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl) trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>175617</td>
			<td>This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818</td>
			<td>Absolute, 200 Proof, molecular biology grade</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>BioShop Canada Inc.</td>
			<td>ALB001</td>
			<td>Often referred to as Cohn fraction V.</td>
		</tr>
		<tr>
			<td>N-g-maleimidobutyryloxy succinimide ester (GMBS)</td>
			<td>Pierce Biotechnology</td>
			<td>22309</td>
			<td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Pierce Biotechnology</td>
			<td>31000</td>
			<td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td>
		</tr>
		<tr>
			<td>Biotinylated mouse anti-human anti-CD63</td>
			<td>Ancell</td>
			<td>215-030</td>
			<td>clone AHN16.1/46-4-5</td>
		</tr>
		<tr>
			<td>biotinylated annexin V</td>
			<td>BD Biosciences</td>
			<td>556418</td>
			<td>Annxin V has a high affinity for phosphotidylserine (PS)</td>
		</tr>
		<tr>
			<td>Primary anti-CD9 and secondary antibody</td>
			<td>System Biosciences</td>
			<td>EXOAB-CD9A-1</td>
			<td>The secondary antibody is horseradish peroxidise-conjugated</td>
		</tr>
		<tr>
			<td>Serum separation tubes</td>
			<td>BD Biosciences</td>
			<td>367991</td>
			<td>Clot activator and gel for serum separation</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer</td>
			<td>BD Biosciences</td>
			<td>556454</td>
			<td>10X; dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>TMB substrate reagent set</td>
			<td>BD Biosciences</td>
			<td>555214</td>
			<td>The set contains hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1560</td>
			<td>MirVana RNA isolation kit</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone-based RNA isolation aid</td>
			<td>Life Technologies</td>
			<td>AM9690</td>
			<td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td>
		</tr>
		<tr>
			<td>RNA cleanup kit</td>
			<td>Qiagen Inc.</td>
			<td>74004</td>
			<td>MinElute RNA cleanup kit is designed for purification of up to 45 &#956;g RNA.</td>
		</tr>
		<tr>
			<td>Plasma chamber</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Hitachi Ltd.</td>
			<td>S-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop scanner</td>
			<td>Hewlett-Packard Company</td>
			<td>Photosmart B110</td>
			<td>8-bit color images were captured. Cameras and smart phones may be also used.</td>
		</tr>
		<tr>
			<td>Image-record system</td>
			<td>J&#38;H Technology Co</td>
			<td>GeneSys G:BOX Chemi-XX8</td>
			<td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td>
		</tr>
	</tbody>
</table>

paper-based devices for isolation and characterization of extracellular vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

The ability of the paper device to isolate subgroups of EVs efficiently relies upon its sensitive and specific recognition of EV surface markers. The stable modification of paper fibers with capture molecules is achieved by using avidin-biotin chemistry as described elsewhere28-30. The effectiveness of chemical conjugation and that of the physisorption method is assessed using fluorescence-based readouts. The paper test zones are prepared following the protocol step 1) except the capture molecule is replaced w...

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

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JoVE Journal

Bioengineering

11.3K Görüntüleme.  National Tsing Hua University.  Bu protokol, 10 kadar ul serum örneklerinden, hücre dışı veziküller (EVS), hücrelerden salınan küçük zar parçacıkları izole etmek için bir yöntem göstermektedir. Bu yaklaşım, ultra-santrifüj ihtiyacını önlediği deney süresi sadece birkaç dakika sürer ve sınırlı hacim örneklerinden EV'lerin izolasyonunu sağlar. 