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.
