This study was supported by the National Research Foundation of Korea, Grant NRF-2018R1D1A1A09084044. J. H. Lee was supported by a research grant from Kwangwoon University in 2019. Hyerin Kim was supported by the &#8220;Competency Development Program for Industry Specialists&#8221; of the Korean Ministry of Trade, Industry and Energy (MOTIE), operated by the Korea Institute for Advancement of Technology (KIAT) (No. P0002397, HRD program for Industrial Convergence of Wearable Smart Devices).

1. Device fabrication
<ol>
	<li>Define the region to be printed on paper using printer software (Table of Materials). 
	NOTE: The design has 12 wax-patterned layers in which the diameters of the circular sample areas gradually narrow from 5 mm to 2 mm (Figure 1A).</li>
	<li>Print hydrophobic wax on the designated regions on both sides of the cellulose paper (Table of Materials) using a commercial wax printer (Table of Materials) (<str...

<ol>
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The authors have nothing to disclose.

Although the Exo-PAD was used successfully for the enrichment and isolation of microvesicles and exosomes, several critical points should be carefully considered: 1) the oven incubation time and temperature during the device preparation, 2) processing time, 3) application of voltage with varying layer numbers and sample area diameters, and 4) applicability to clinical samples.
The incubation time and temperature given in the protocol are optimized conditions to fabricate a reliable device. Lon...

Microvesicles and exosomes are small membrane vesicles measuring 0.2&#8722;1 &#956;m and 30&#8722;200 nm, respectively. They are secreted into the extracellular environment by several different cell types1,2,3,4,5. They contain parental cell information in the form of subsets of DNA, mRNA, miRNA, proteins, and lipids, and circulate throughout the body via various body fluids such as serum, plasma, urine, cerebrospinal fluid, amniotic fluid, and saliva6,7,8,9. Thus, techniques for efficient isolation of microvesicles and exosomes from biological fluids can provide extensive opportunities in the fields of the diagnosis, prognosis, and real-time monitoring of disease, as well as in the development of new therapeutics.
However, the conventional isolation method for microvesicles and exosomes based on ultracentrifugation is extremely time-consuming and causes significant loss and contamination of the sample. This is because it involves several cumbersome pipetting and loading steps and discarding of various reagents with repeated ultracentrifugation5,6,10,11,12. Moreover, the high shear stress induced by ultracentrifugation (~100,000 x g) can cause the physical lysis of microvesicles and exosomes, yielding poor recovery rates (5&#8722;23%)6,13,14. Therefore, a highly efficient, unobtrusive isolation technique for microvesicles and exosomes must be developed to reduce damage and loss, thereby achieving higher recovery rates.
An origami-paper-based device (Exo-PAD) was developed for simpler, gentler, and highly efficient isolation of microvesicles and exosomes6. The design of the Exo-PAD is a multifolded paper with serially connected sample areas that gradually decrease in diameter. The ion concentration polarization (ICP) technique, which is a nano-electrokinetic phenomenon that preconcentrates charged biomolecules, was integrated with this unique design. Using the Exo-PAD resulted in fivefold enrichment of the microvesicles and exosomes in specific layers and their isolation by simply unfolding the device. This article describes the Exo-PAD in detail, from the overall device fabrication and operation to analysis of its use, to illustrate the method and show representative results6.

<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

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

The operation time must be optimized to achieve the maximum recovery yield of the enriched microvesicles and exosomes. Insufficient time does not allow sufficient migration of the microvesicles and exosomes, which decreases the enrichment, whereas excessive time deteriorates the spatial focusing and hence disperses the microvesicles and exosomes. Thus, through the time optimization step, the maximum preconcentration factor of microvesicles and exosomes and the final location where microvesicles and exosomes are most enri...

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

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

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

In this protocol, we describe a simple method to fabricate a paper-based device called the XO Pad, which is designed for the effective enrichment in isolation of extracellular vesicles. The XO Pad is a simple device that can pre-concentrate and isolate extracellular vesicle without the use of any chemicals or working machines, while showing higher recovery rates than conventional methods. Begin by defining the region to be printed with printer software.The design should have 12 wax patterned layers in which the diameters of the circular sample areas gradually narrow from five to two millimeters. Then, print hydrophobic wax on the designated regions on both sides of the cellulose paper. Place the wax printed paper in a laboratory oven for 80 seconds at 120 degrees Celsius, then cut the wax printed paper with a cutter to make individual XO Pad devices.Drop a five and two microliters of perm selective membrane onto the sample areas in the left most and right most layers, respectively. Place the layers coded with the perm selective membrane on a hot plate at 70 degrees Celsius for 30 minutes to evaporate the solvent. Then seal the outermost surface of the coated layer facing the buffer solution with pressure sensitive tape, leaving a small hole.Fold the XO Pad device back and forth along the white lines to convergently connect all sample areas. Pipette 15 microliters of the microvesicle and exosome sample in the convergent sample areas and wait a few seconds to ensure complete wetting. Place two acrylic chambers at both ends of the XO Pad and clamp it securely with small binder clips to prevent unfolding.Fill the chambers with 110 microliters of 0.1X PBS and insert two silver electrodes. Then use a current voltage source measurement system to apply 30 volts to the electrodes for 20 minutes. To isolate the enriched microvesicles and exosomes, separate the folded XO Pad from the acrylic chambers and unfold the device to isolate the enriched particles from the other layers.Punch out the sample areas in layers eight and nine where the microvesicles and exosomes are enriched for downstream analysis. A time-lapse migration assay of fluorescently labeled microvesicles and exosomes was performed in order to optimize the operation time for maximum recovery of the enriched particles. The fluorescence intensities in all sample areas were quantified with ImageJ software.Before the enrichment process, microvesicles and exosomes were dispersed in all sample areas with a gradual decrease in the intensity. After 10 minutes of processing, the particles migrated electro-kinetically and an instant pre-concentration plug appeared on layer seven. When the time reached 20 minutes, microvesicles and exosomes were strongly focused on layers eight and nine and enriched five fold based on the fluorescence intensities.The integrity of the enriched microvesicles and exosomes in layer eight was investigated with SEM. Significantly more non-damaged particles were observed after the enrichment process. The size distribution of the enriched microvesicles and exosomes was also analyzed.Subsequently, the actual concentration of the enriched microvesicles and exosomes on the sample areas in layers six, eight, and 10 was analyzed. In layer eight, the microvesicles and exosomes were pre-concentrated by a factor of 5.58, but no noticeable pre-concentration was observed in the other layers. To ensure the reproducibility of this procedure, precisely followed the protocol for oven incubation time and temperature during device verification and device operation time because those are the optimized conditions.The XO Pad can also be used to separate and isolate extracellular vesicle from protein-rich samples, such as serum or plasma, by using carbonate buffer. This protocol can provide an efficient, simple preparation tool, with its pre-concentrating and isolating capabilities required for clinical research, such as diagnosis, drug delivery, immunotherapy, and other therapeutics.

paper-based-preconcentration-isolation-microvesicles

Research

JoVE Journal

Biochemistry

5.9K visualizações.  Kwangwoon University. Neste protocolo, descrevemos um método simples para fabricar um dispositivo baseado em papel chamado XO Pad, que foi projetado para o enriquecimento eficaz no isolamento de vesículas extracelulares. O XO Pad é um dispositivo simples que pode pré-concentrar e isolar vesícula extracelular sem o uso de quaisquer produtos químicos ou máquinas de trabalho, ao mesmo tempo em que mostra taxas de recuperação mais altas do que os métodos convencionais. Comece definindo a região a ser impres...