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 paper pieces with two polystyrene sheets with registered through-holes, and laminate.</li>
	<li>Modify paper devices using the chemical conjugation method described in the following steps28.
	<ol>
		<li>Treat paper devices with oxygen plasma (100 mW, 1% oxygen, 30 sec) in a plasma chamber. 
		CAUTION: Special caution must be taken when working with 3-mercaptopropyl trimethoxysilane and N-&#947;-maleimidobutyryloxy succinimide ester (GMBS), since they are both moisture sensitive and toxic. Wear protective gloves and avoid inhalation or contact with skin and eyes. Keep the stock bottle tightly closed and allow it to equilibrate to RT before opening to avoid water condensation. Alternatively, open the stock bottle inside a nitrogen-filled glove bag or box. Aliquot into small vials to avoid frequent opening of the stock bottle.</li>
		<li>Immediately incubate treated paper devices in a 4% (v/v) solution of 3-mercaptopropyl trimethoxysilane in ethanol (200 proof) for 30 min.</li>
		<li>Rinse paper devices with ethanol and incubate them with 0.01 &#956;mol/ml GMBS in ethanol for 15 min.</li>
		<li>Rinse with ethanol (200 proof) and incubate paper devices with 10 &#181;g/ml avidin solution in phosphate buffered saline (PBS) for 1 hr at 4 &#176;C. Store at 4 &#176;C if needed, and use within 4 weeks.</li>
	</ol>
	</li>
	<li>Wet each paper test zones with 10 &#956;l PBS containing 1% (w/v) bovine serum albumin (BSA) for 3 &#215; 10 min before spotting 10 &#956;l biotinylated capture molecules for 3 &#215; 10 min. Use anti-CD63 antibody (20 &#956;g/ml in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide) or annexin V (1:20, v/v in annexin V binding buffer) as capture molecules.</li>
	<li>Rinse off unbound anti-CD63 antibody or annexin V molecules using 10 &#956;l corresponding PBS containing 1% (w/v) BSA or annexin V binding buffer solutions for 3 &#215; 1 min, respectively.</li>
</ol>
2. Serum and Aqueous Humor Sample Collection and Processing
<ol>
	<li>Serum collection.
	<ol>
		<li>Collect 10 ml of peripheral blood by venipuncture in serum separation tubes and gently invert the tube five times. Set the tube in a vertical position and wait for 30 min.</li>
		<li>Centrifuge at 1,200 &#215; g for 15 min. Transfer the serum from the top layer to a clean tube, and centrifuge again at 3,000 &#215; g for 30 min.</li>
		<li>Pass the supernatant through a 0.8 &#956;m filter. Keep the sample at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Aqueous humor collection: collect aqueous humor samples from patients diagnosed by an ophthalmologist directly through invasive procedures and store samples at -80 &#176;C until use.</li>
</ol>
3. Isolation of Extracellular Vesicles
<ol>
	<li>Spot samples onto each paper test zone at a rate of 5 &#956;l/min.</li>
	<li>Rinse off unbound EVs with 10 &#956;l corresponding PBS containing 1% (w/v) BSA or annexin V binding buffer solutions for 3 &#215; 1 min for paper devices functionalized with anti-CD63 antibody or annexin V molecules, respectively. Perform the following downstream assays.</li>
</ol>
4. Downstream Assay Example 1: Scanning Electromicrographs
<ol>
	<li>Fix EVs captured on functionalized paper test zone using 10 &#956;l 0.5&#215; Karnovsky&#8217;s fixative for 10 min.</li>
	<li>Rinse the samples with ample PBS for 2 &#215; 5 min.</li>
	<li>Dehydrate the samples with subsequent 35% ethanol for 10 min, 50% ethanol for 2 &#215; 10 min, 70% ethanol for 2 &#215; 10 min, 95% ethanol for 2 &#215; 10 min, and 100% ethanol for 4 &#215; 10 min.</li>
	<li>Critical dry and sputter coat the samples with palladium/gold, and examine using an scanning electron microscope operated at low electron acceleration voltage (~ 5 kV).</li>
</ol>
5. Downstream Assay Example 2: Paper-based ELISA
<ol>
	<li>Add a 5 &#956;l solution containing anti-CD9 primary antibody at 1:1,000 dilution in PBS to each test zone containing captured EVs. Wait 1&#160; min.</li>
	<li>Rinse the samples with 30 ml PBS shaken at 100 rpm for 30 sec.</li>
	<li>Add a 5 &#956;l solution containing horseradish peroxidase (HRP)-linked secondary antibody at 1:1,000 dilution in PBS and wait 1 min.</li>
	<li>Rinse the samples with 30 ml PBS shaken at 100 rpm for 30 sec.</li>
	<li>Add a 5 &#956;l colorimetric substrate containing 1:1 volume ratio of hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB) and scan using a desktop scanner.</li>
</ol>
6. Downstream Assay Example 3: RNA Isolation
<ol>
	<li>Immerse the samples in 35 &#956;l of polyvinylpyrrolidone-based RNA isolation aid and 265 &#956;l of lysis/binding buffer to lyse EVs captured. Vortex vigorously.</li>
	<li>Add 30 &#956;l homogenate provided in the isolation kit to the lysate. Vortex vigorously and put on ice for 10 min.</li>
	<li>Extract the RNA using acid phenol-chloroform separation described in the following steps.
	<ol>
		<li>Take 330 &#956;l of phenol-chloroform from the bottom layer of the bottle and add to the homogenate/lysate. Vortex vigorously.</li>
		<li>Centrifuge at 10,000 x g for 5 min. Transfer the aqueous (upper) phase to a clean tube and note the volume removed.</li>
	</ol>
	</li>
	<li>Precipitate the total RNA with 100% ethanol of the volume that is 1.25 times the volume of the aqueous phase removed in the previous step and collect the RNA in the elution solution preheated to 95 &#176;C.</li>
	<li>Concentrate and further purify the RNA using an RNA cleanup kit according to the manufacture&#8217;s protocol.</li>
</ol>

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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 fluids. We chose chromatography cellulose paper grade 1 mainly for its proper pore size and low protein adsorption. Compared to another commonly used porous materials, nitrocellulose, cellulose paper has much lower nonspecific protein adsorption34. We also find little nonspecific capture of EVs in this study. In addition, its particle retention level, 11 &#956;m, is larger than the size of EVs35, allowing for specific capture in contrast to physical trapping of EVs. Here, we focus on EVs of a smaller size by passing serum samples through a 0.8 &#956;m filter, which step may be modified when larger EVs are to be studied.
Chemical surface modification can be a key to achieve a stable and high coverage of capture molecules on the surface as a means of improving the efficiency of EV isolation. Surface modifications of cellulose have been reviewed36,37. There are high numbers of hydroxyl (-OH) groups on the surface of cellulose and they can react with silane to give stable chemical Si-O-C bonds after being activated with oxygen plasma or other condensation process. The silane used in this study is (3-mercaptopropyl) trimethoxysilane, (MeO)3Si-(CH2)3-SH. The thiol (-SH) group can react with GMBS to provide a short space arm (0.73 nm) and a crosslinking group that couples with primary amines. Capture molecules with amine groups can be conjugated to GMBS directly. In this study, however, we conjugated NeutrAvidin instead to provide a general scheme to conjugate various capture molecules that are biotin-labeled. However, consensus on EV subtype-specific markers has yet to be reached17. NeutrAvidin has a very strong affinity to biotin and a near-neutral isoelectric point (pH 6.3), minimizing nonspecific interactions with negatively charged objects, e.g. the surface of EVs. In contrast, avidin with an isoelectric point of 10.5 carries positive charges at neutral pH. A higher concentration of anti-CD63 antibody is used than that in Chen et al.20, mainly due to the small volume applied and the larger surface-to-volume ratio of paper device. However, the amount and compositions of proteins adsorbed onto the paper fibers have not been characterized, which may be sample-dependent. Hence, care must be taken when interpreting the results of protein analysis.
Collected biological samples should be handled gently and processed rapidly to prevent artifactually increased EV levels. It is recommended to remove cells and platelets, if any, before storage at -80 &#176;C. A current standardization of sample handling in EV research can be found in this review17.
Proper laboratory hygiene practice should be employed. Always wear gloves and change gloves frequently to minimize the introduction of dust particles and ribonucleases. Isolation of RNA from EVs in cell culture-conditioned media can yield more than 70 times greater concentrations using paper devices as compared to that extracted from EVs concentrated using the ultracentrifuge method (unpublished).
The protocol described herein uses cellulose paper devices modified chemically with capture molecules to isolate different subgroups of EVs. The method is simple, efficient, and especially valuable when the sample volume is limited.

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 with 20 &#956;g/ml fluorophore R-phycoerythrin-conjugated biotin molecules (PE-biotin) in step 1.3. The paper test zones are then rinsed with PBS to remove unbound PE-biotin molecules. In contrast, 10 &#956;l 20 &#956;g/ml PE-biotin in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide is directly spotted on the paper test zone for 3 &#215; 10 min, followed by PBS rinse for conducting the physisorption of PE-biotin dye molecules. Chemically modified paper devices (&#34;paper+crosslinker+dye&#34;) along with unprocessed paper devices (&#34;paper&#34;), paper devices modified with the same protocol without the application of PE-biotin molecules (&#34;paper+crosslinker&#34;) and paper devices with physisorbed PE-biotin molecules (&#34;physisorbed-dye&#34;) are scanned using an image-record system set to 540-560 nm excitation, 575 nm-long-pass emission, and the data is saved in the form of 16-bit files and analyzed using ImageJ software. As shown in Figure 2A, fluorescence intensity of test zones prepared by the chemical conjugation method is substantially higher, as compared to physically coated paper test zones (p-value &#60; 0.0005, n = 5, Student&#8217;s t-test). The capturing of EVs is also specific as exemplified in Figure 2B, C, where few EVs are retained on the paper device when the capture molecules are replaced by PBS solution.
EVs are heterogeneous in sizes, specific contents, and biogenesis routes. To bind them, paper devices coated with two different EV capture molecules, anti-CD63 antibodies and annexin V, were prepared. CD63, a member of the tetraspanin family (which includes CD9, CD63, CD81 and CD82 molecules) is enriched on EV membrane31, and annexin V has a strong affinity for phosphatidylserine (PS)32,33. It is found that EVs are released constitutively during early apoptosis or under stress conditions, in which process normal cell phospholipid asymmetry is lost, leading to increased exposure of PS on the outer leaflet of the EV membrane. Analyses of SEM images show that the size distributions of CD63+ EVs and PS+ EVs are different, with mean diameters of 80 and 43 nm, respectively (Figure 3).The two-tailed Student&#8217;s t-test is used to determine the statistical p-value (p-value &#60; 0.0001). Surprisingly, CD63+ EVs appear rounder and larger than PS+ EVs on average, which biological significance remains to be elucidated.
RNA from EVs captured on the paper device can be extracted. Analyses with a bioanalyzer showed similar overall profiles for total RNAs extracted from CD63+ and PS+ EVs (Figure 4A). Compared to the amounts of RNA extracted using the microfluidic and ultracentrifugation techniques, respectively about twice and 39 times more RNAs per unit volume of serum sample were extracted utilizing the paper device, albeit using a smaller sample volume (Table 1). P-ELISA can be carried out for the detection of EVs. Anti-CD9 primary antibody and HRP-conjugated secondary antibody are used in this study. Figure 4B displays grey value changes produced by the enzymatic reaction of HRP for paper devices applied with 10 &#956;l PBS, 50% serum (diluted with PBS), or 100% serum, respectively. The grey value increases linearly within the range of serum samples tested.
Paper devices are especially advantageous when the sample amount is limited such as in the case of aqueous humor, when only &#60; 200 &#956;l/eye can be collected. Multiple samples are often needed and pooled together if other isolation methods, e.g., ultracentrifugation, are to be used. EVs from patient aqueous humor samples isolated with paper devices coated with anti-CD63 antibodies can be seen in SEM images as showed in Figure 5.
<img alt="Figure 1" src="/files/ftp_upload/52722/52722fig1.jpg" /> 
Figure 1. The fabrication and operation procedure of paper-based assays aimed at isolation and characterization of extracellular vesicles.&#160;(A) Filter paper is cut into the desired shape and laminated. (B) The surface of the paper is modified with a brief treatment of oxygen plasma and reacted with 3-mercaptopropyl trimethoxysilane, N-&#947;-maleimidobutyryloxy succinimide ester, and NeutrAvidin, in that order. Biotinylated capture molecules, e.g., antibodies, can then be immobilized via the strong affinity between biotin and NeutrAvidin molecules. (C) Biosamples can be pipette onto the functionalized paper device. Extracellular vesicles can be isolated. (D) Downstream assays, such as SEM, transcriptome, and ELISA (shown), can be performed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52722/52722fig2.jpg" /> 
Figure 2. Extracellular vesicles (EVs) can be captured on functionalized paper devices with good specificity and sensitivity.&#160;(A) Fluorescently labeled biotin molecules are highly immobilized onto the paper device via chemical conjugation process (&#34;paper+crosslinker+dye&#34;) in contrast to physical adsorption (&#34;physisorbed-dye&#34;). Unprocessed paper (&#34;paper&#34;) and conjugation molecules (&#34;paper+crosslinker&#34;) contribute little to the fluorescence intensity. (B) A representative SEM image showing few EVs are captured on the paper device non-specifically when the capture molecules is replaced by PBS containing 1% (w/v) BSA. (C) Many EVs are retained on paper devices coated with anti-CD63 antibodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3-mercaptopropyl" src="/files/ftp_upload/52722/52722fig3.jpg" /> 
Figure 3. Subgroups of Extracellular vesicles (EVs) can have different size distributions.&#160;(A) and (B) are representative SEM images of EVs captured on paper devices coated with anti-CD63 antibodies and annexin V molecules, respectively. (C) CD63+ EVs appear larger than PS+ EVs under experimental conditions used in this study (p-value &#60; 0.0001, n = 124 and 203, Student&#8217;s t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52722/52722fig4.jpg" /> 
Figure 4. Downstream transcriptome and ELISA assays. (A) Bioanalyzer data showing the intensities (y-axis, in arbitrary units) and size (x-axis, in nucleotides (nt)) distributions of fluorescently-labeled total RNA extracted from EVs captured on anti-CD63 antibody- or annexin V-coated filter papers. The migrating peak at ~25 nt represents an internal standard. (B) Grey value changes produced by the enzymatic reaction of horseradish peroxidase (HRP) in the P-ELISA assay for paper devices applied with 10 &#956;l samples of PBS, 50% serum (diluted with PBS) and 100% serum samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52722/52722fig5.jpg" /> 
Figure 5. Extracellular vesicles (EVs) in aqueous humor samples can be captured on functionalized filter paper without sample pooling. SEM images of isolated CD63+ EVs in aqueous humor samples from patients with (A) age-related macular degeneration, (B) diabetic macular edema, and (C) polypoidal choroidal vasculopathy, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>isolation method</td>
			<td>capture molecule</td>
			<td>serum vol. (&#181;l)</td>
			<td>average RNA extracted (ng)</td>
			<td>average extracted RNA/serum 
			(pg/&#181;l)</td>
			<td>ratio of average RNA/serum</td>
			<td>ref</td>
		</tr>
		<tr>
			<td>microfluidic device</td>
			<td>anti-human CD63</td>
			<td>400</td>
			<td>4.14</td>
			<td>10.4</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>paper device</td>
			<td>anti-human CD63</td>
			<td>72</td>
			<td>1.49 &#177; 0.08</td>
			<td>20.7</td>
			<td>39</td>
			<td>22</td>
		</tr>
		<tr>
			<td>paper device</td>
			<td>annexin V</td>
			<td>72</td>
			<td>0.99 &#177; 0.26</td>
			<td>13.8</td>
			<td>26</td>
			<td>22</td>
		</tr>
		<tr>
			<td>ultracentrifugation</td>
			<td>-</td>
			<td>2,000</td>
			<td>1.06 &#177; 0.64</td>
			<td>0.53</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>serum</td>
			<td>-</td>
			<td>72</td>
			<td>1.23 &#177; 0.58</td>
			<td>17</td>
			<td>32</td>
			<td>22</td>
		</tr>
	</tbody>
</table>
Table 1. Total RNA extracted from serum samples from healthy volunteers analyzed with a bioanalyzer (n = 4 for all conditions).

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 ...

paper-based-devices-for-isolation-characterization-extracellular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.3K Views.  National Tsing Hua University. This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 μl 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.

Video: Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

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Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...