SIZE-SELECTIVE ISOLATION OF URINARY EXOSOME

ON LAB-ON-A-DISC

Hyun-Kyung Woo1, Ja-Ryoung Han1, Vijaya Sunkara1, and Yoon-Kyoung Cho*1,2

1Department of Biomedical Engineering, Ulsan National Institute of Science and Technology

(UNIST) UNIST-gil 50, Ulsan 689-798, South Korea 2Center for Soft and Living Matter, Institute for Basic Science (IBS) UNIST-gil 50, Ulsan 689-

798, South Korea

ABSTRACT

A size selective isolation of exosome is demonstrated on a centrifugal microfluidic system.

Starting with 1 mL of raw urine sample, we suggest a rapid and easy method for isolation of exosomes by

using nanosized filters integrated on a lab-on-a-disc. Our method is a cost effective, quick, and painless

method for identification of the bacterium by analyzing the RNA in the exosomes secreted in the

patient’s urine. It may be used routinely for early diagnostics of hospitalized patients susceptible to sepsis,

dramatically lowering its mortality rate.

KEYWORDS: Exosome, Isolation, Sepsis, Lab-on-a-disc

INTRODUCTION

Millions of people throughout the world suffer from sepsis which mortality rates are as high as

30%, making it the most common cause of death in hospitalized patients. Sepsis infection is most com-

monly caused by bacteria, and the causative organism infected is currently identified by obtaining at least

two sets blood cultures from the patient. We offer a cost effective, quick, and painless method for

identification of the bacterium by analyzing the RNA in the exosomes secreted in the patient’s

urine. To note, this exosome is not to be confused with the commonly known molecular machine that

degrades RNA. They are 40-120 nm in size, contain RNA, and are secreted from cells in the form of ves-

icles [1]. We selected exosomes as a candidate based on its communication between bacterial pathogen

and host cells, and that it contains information of both gram-negative and -positive bacteria infecting the

host in the form of RNA. Currently, ultracentrifugation is commonly used to isolate exosomes, but it re-

quires a large sample volume (~50 mL) and a long time (~4 hours). To overcome this limitation, there are

other approaches such as immune-isolation, and filtration methods using precipitating reagent. Immuno-

isolation method has high selectivity, but it is costly and inefficient because of its use of antibodies. In

the case of isolation with precipitating reagents providing high purity, this method requires a long time

Figure 1. A schematic view of a disc and its principle. (a) The device is consisted of 4 chambers and three wax

valves. (b) The filter was equilibrated prior to running the samples, and the waste channel was located so that the

buffer can fill and wet the whole filter. (c) Two different size filters, 600 nm and 30 nm, were used to isolate exo-

somes, and (d) Nanobeads sized with 100 nm and 800 nm were mixed and applied to verify that only the 100 nm

beads were excluded.

463978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA

with several centrifugation steps [2]. To achieve better results over these limitations, we suggest a size-

selective method using nanosize filters integrated on a lab-on-a-disc platform.

EXPERIMENTAL

Figure 1(a) illustrates the schematic of the device. A disc consisted of 4 chambers and three wax

valves. The details of the fabrication can be found elsewhere [3]. Figure 1(b) and (c) shows its core me-

chanics. Two different size filters, 600 nm and 30 nm, were used to isolate exosomes. To demonstrate

disc performance, nanobeads sized with 100 nm and 800 nm were mixed thoroughly and put in to the fil-

ter chamber and only the 100 nm sized bead particles were excluded as shown in Figure 1(d).

RESULTS AND DISCUSSION

The fluidic operation of the disc is shown in Figure 2, 1 mL of the sample (red) are introduced to

the sample chamber. Keeping the valve #1 closed, the debris of the sample chamber (red) is precipitated

due to centrifugal force (Figure 2(a)) while PBS (yellow) runs from its chamber to equilibrate the filter

(Figure 2(b)). After PBS chamber is emptied, valve #2 must be closed to prevent back flow (Figure 2(c)).

Opening valve #1, the sample drains to the filter chamber and further to the waste chamber by centrifugal

force (Figure 2(d)). Valve #3 is closed and valve #2 is reopened to wash the filter (Figure 2(e)). The filter

is washed twice with PBS (colorless solution). The 30 nm filter is then removed to collect the isolated

exosomes. The fully automated process takes about 20 minutes.

The exosomes were detected by transmission electron microscope (TEM) to confirm its structure

and size (Figure 3(a)). The structure of the exosome isolated by the disc, acquired from both healthy sub-

ject (left) and the sepsis patient (middle), was consistent with that acquired by ultracentrifugation (right).

Figure 2. Demonstration of the fluidic operation on a spinning disc. Colored liquid was used for easy visualization

of the fluidic transfer on the disc. (a) Keeping the valve #1 closed, the debris of the sample chamber (red) is precip-

itated due to centrifugal force while (b) PBS (yellow) runs from its chamber to equilibrate the filter. (c) After the

PBS chamber has emptied, valve #2 is closed to prevent back flow. (d) Opening valve #1, the sample drains to the

filter chamber and further to the waste chamber by centrifugal force. (e) Valve #3 is closed and valve #2 is reo-

pened to wash the filter, and (f) The filter is washed twice with PBS (colorless solution). The 30 nm filter is then

removed to collect the isolated exosomes.

464

All exosomes were confirmed to have the size between 40 and 120 nanometers. The experiments with

known concentration of E. coli exosome was shown to have 80% recovery (Figure 4(b)), and the RNA in

the exosome were confirmed to be intact by reverse transcriptase-PCR (Figure 4(c)).

CONCLUSION

Overall, we demonstrated the time effective, reasonable cost and miniaturized system which re-

quires only a small sample volume. Our compact and miniaturized platform could highly contribute for

early diagnosis of hospitalized patients susceptible to sepsis, dramatically lowering its mortality rate.

ACKNOWLEDGEMENTS

This work was supported by National Research Foundation (NRF) grant

(2013R1A2A2A05004314) and a grant from the Korean Health Technology R&D Project, Ministry of

Health & Welfare (A121994), and IBS-R020-D1 funded by the Korean Government.

REFERENCES

[1] S.E. Andaloussi et al., “Extracellular vesicles: biology and emerging therapeutic opportunities,” Na-

ture Review, 12, 347, 2013.

[2] R.E. Lane et al., “Analysis of exosome purification methods using a model liposome system and

tunable-resistive pulse sensing,” Scientific Reports, 5, 7639, 2015.

[3] T.H. Kim et al., “A Fully Integrated Lab-on-a-Disc for Nucleic Acid Analysis of Food-borne Patho-

gens,” Analytical Chemistry, 86, 3841, 2014.

CONTACT

*Y. K. Cho, Tel: +82-052-217-2511; E-mail: ykcho@unist.ac.kr, http://fruits.unist.ac.kr

Figure 3. Physical and surface characterization of exosome isolated on a disc. (a) Transmission electron microscope

(TEM) images of isolated exosome from the urine of a healthy subject (left), and patient (middle) using the disc,

and a positive sample from cultured media (right), (b) The recovery rate based on BCA analysis and (c) PCR for

16SrRNA was confirmed with exosome samples prepared by the lab-on-a-disc.

465