Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Summary

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

Abstract

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  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.

Introduction

Microvesicles and exosomes are small membrane vesicles measuring 0.2−1 μm and 30−200 nm, respectively. They are secreted into the extracellular environment by several different cell types^1,2,3,4,5. 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 saliva^6,7<....

Protocol

1. Device fabrication

1. 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).
2. 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) (Figure 1B).
3. Place the wax-printed paper in a laboratory oven for 80 s at 120 °C.
   NOTE: This step allow....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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