High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis
Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc)...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2024
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/174668 |
| _version_ | 1826120420979376128 |
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| author | Leong, Sheng Yuan Lok, Wan Wei Goh, Kah Yee Ong, Hong Boon Tay, Hui Min Su, Chengxun Kong, Fang Upadya, Megha Wang, Wei Radnaa, Enkhtuya Menon, Ramkumar Dao, Ming Dalan, Rinkoo Suresh, Subra Lim, Darren Wan-Teck Hou, Han Wei |
| author2 | School of Mechanical and Aerospace Engineering |
| author_facet | School of Mechanical and Aerospace Engineering Leong, Sheng Yuan Lok, Wan Wei Goh, Kah Yee Ong, Hong Boon Tay, Hui Min Su, Chengxun Kong, Fang Upadya, Megha Wang, Wei Radnaa, Enkhtuya Menon, Ramkumar Dao, Ming Dalan, Rinkoo Suresh, Subra Lim, Darren Wan-Teck Hou, Han Wei |
| author_sort | Leong, Sheng Yuan |
| collection | NTU |
| description | Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects (n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing. |
| first_indexed | 2024-10-01T05:16:11Z |
| format | Journal Article |
| id | ntu-10356/174668 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:16:11Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | ntu-10356/1746682024-04-23T03:11:40Z High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis Leong, Sheng Yuan Lok, Wan Wei Goh, Kah Yee Ong, Hong Boon Tay, Hui Min Su, Chengxun Kong, Fang Upadya, Megha Wang, Wei Radnaa, Enkhtuya Menon, Ramkumar Dao, Ming Dalan, Rinkoo Suresh, Subra Lim, Darren Wan-Teck Hou, Han Wei School of Mechanical and Aerospace Engineering School of Biological Sciences School of Materials Science and Engineering Lee Kong Chian School of Medicine (LKCMedicine) Tan Tock Seng Hospital Engineering Platelet-free plasma Liquid biopsy Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects (n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing. Nanyang Technological University National Medical Research Council (NMRC) Submitted/Accepted version H.W.H. would like to acknowledge the financial support from NTUitive POC Gap Funding (NGF-2020-09-010), SMART Ignition Grant (ING-001058BIOIGN), and NTU Start up grant. S.Y.L. would like to acknowledge support from the NTU Research Scholarship. D.W.-T.L. and K.Y.G. are supported by NMRC/CSAINV/0025/2017. 2024-04-07T06:43:05Z 2024-04-07T06:43:05Z 2024 Journal Article Leong, S. Y., Lok, W. W., Goh, K. Y., Ong, H. B., Tay, H. M., Su, C., Kong, F., Upadya, M., Wang, W., Radnaa, E., Menon, R., Dao, M., Dalan, R., Suresh, S., Lim, D. W. & Hou, H. W. (2024). High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis. ACS Nano, 18(8), 6623-6637. https://dx.doi.org/10.1021/acsnano.3c12862 1936-0851 https://hdl.handle.net/10356/174668 10.1021/acsnano.3c12862 38348825 2-s2.0-85185580524 8 18 6623 6637 en NGF-2020-09-010 ING-001058BIOIGN NTU-SUG NMRC/CSAINV/0025/2017 ACS Nano 10.21979/N9/VGLHMU © 2024 American Chemical Society. All rights reserved. This article may be downloaded for personal use only. Any other use requires prior permission of the copyright holder. The Version of Record is available online at http://doi.org/10.1021/acsnano.3c12862. application/pdf application/pdf |
| spellingShingle | Engineering Platelet-free plasma Liquid biopsy Leong, Sheng Yuan Lok, Wan Wei Goh, Kah Yee Ong, Hong Boon Tay, Hui Min Su, Chengxun Kong, Fang Upadya, Megha Wang, Wei Radnaa, Enkhtuya Menon, Ramkumar Dao, Ming Dalan, Rinkoo Suresh, Subra Lim, Darren Wan-Teck Hou, Han Wei High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis |
| title | High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis |
| title_full | High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis |
| title_fullStr | High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis |
| title_full_unstemmed | High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis |
| title_short | High-throughput microfluidic extraction of platelet-free plasma for MicroRNA and extracellular vesicle analysis |
| title_sort | high throughput microfluidic extraction of platelet free plasma for microrna and extracellular vesicle analysis |
| topic | Engineering Platelet-free plasma Liquid biopsy |
| url | https://hdl.handle.net/10356/174668 |
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