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Submicron particle focusing and exosome sorting by wavy microchannel structures within viscoelastic fluids Yinning Zhou, Zhichao Ma, Mahnoush Tayebi, and Ye Ai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05749 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019
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Analytical Chemistry
Submicron particle focusing and exosome sorting by wavy microchannel structures within viscoelastic fluids Yinning Zhou, Zhichao Ma, Mahnoush Tayebi and Ye Ai* Pillar of Engineering Product Development, Singapore University of Technology and Design, Singapore 487372, Singapore
* Corresponding author. Email:
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Abstract Exosomes, submicron membrane vesicles (30-200 nm) secreted by almost all cells, containing significant information such as proteins, microRNAs and DNAs, are closely associated with disease diagnostic and prognostic test for liquid biopsy in clinical practice. However, their inherently small sizes lead to great challenges for isolating them from complex body fluids with high throughput and high purity. In this work, a reverse wavy channel structure using viscoelastic fluids with the addition of biocompatible polymer was presented for elasto-inertial focusing and sorting of submicron particles and exosomes. The microfluidic periodically reversed Dean secondary flow generated by repeated wavy channel structures could facilitate particle focusing compared with traditional straight channels. Four differently sized fluorescent submicron spheres (1 µm, 500 nm, 300 nm and 100 nm) were used to study the focusing behavior under various conditions. We have achieved simple, high-throughput and label-free sorting of exosomes with purity higher than 92% and recovery higher than 81%. This developed elasto-inertial exosome sorting technique may provide a promising platform in various exosome-related biological research and pharmaceutical applications.
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Analytical Chemistry
Introduction During the past few decades, liquid biopsy has shown great potentials to become a promising routine test in clinical diagnostic and prognostic detection due to its simple and non-invasive merits alternative to surgical biopsies. For cancer studies and diagnosis, circulating tumor cells (CTCs) and exosomes are quite appealing to researchers because of their comprehensive information contained from the metastatic carcinoma. Exosomes (30-200 nm) are typically cell-derived vesicles secreted into extracellular space, possessing significant information such as surface protein markers, messenger RNAs (mRNAs), microRNAs and DNAs, which can directly reflect the real situation of originated cells and play crucial roles in intercellular communication1-3. Other extracellular vesicles include apoptotic bodies (1-5 µm) secreted by apoptotic cells and microvesicles (100 nm-1 µm) appeared due to conveying progress of substances happened on plasma membrane4,5. Compared with the rare amount of CTCs existing in patient’s peripheral blood (10-100 CTCs per ml6), exosomes have an edge on high concentration not only in peripheral blood, also appear in saliva, urine and synovial fluid etc., exhibiting a more convenient platform for clinical sample obtention7,8. Current exosome isolation methods basically can be classified as size-based and surface marker-based methods9. For size-based approaches, ultracentrifugation has been the gold standard in exosome isolation while it requires super-high speed (＞106 g) and an extremely long processing time, also exhibits the low efficiency and selectivity of exosome recovery10. Although precipitation method can achieve a higher outcome and purity of exosome isolation compared with ultracentrifugation, the addition of precipitation reagents may cause 3 ACS Paragon Plus Environment
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inaccurate analysis on the biological characteristics of isolated exosomes11. Filtration methods utilizing nanoporous membranes can process a large amount of samples but always suffer from clogging issues9,12. For immunoaffinity-based exosome isolation methods normally in conjunction with microfluidic technologies, specific target surface markers (CD63, CD 24, EpCAM and CD41 etc.13,14) are utilized to combine with special microfluidic channel design, such as functionalized nanostructures on gold surfaces15, herringbone groves16 and nanohole arrays17; Other microfluidic approaches have also been developed and demonstrated
for
exosome
isolation
like
membrane-based
filtration
driven
by
electrophoresis18 and pressure19; novel methods with acoustics20, deterministic lateral displacement (DLD)21 and nanowire trapping22. However, most of these approaches normally can only achieve sample throughput lower than 13 µl/min23. It is well-known that inertial microfluidics typically making use of inertial lift force and Dean drag force can effectively manipulate particles larger than 3 µm24,25 with low cost, simplicity and high throughput26. However, when the particle sizes further reduce to the submicron/nanometer scale, the inertial focusing becomes very difficult to achieve as the inertial lift force becomes trivial in this size regime under low pressures (
