Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids
Exosomes are 30–150 nm extracellular vesicles that play crucial roles in intercellular communication and hold significant potential as biomarkers for non-invasive liquid biopsy. However, the current isolation methods have limitations including being time-consuming, producing low yields, and having h...
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MDPI AG
2025-05-01
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| author | Heejong Shin Eunju Jeong Seunggwan Lee |
| author_facet | Heejong Shin Eunju Jeong Seunggwan Lee |
| author_sort | Heejong Shin |
| collection | DOAJ |
| description | Exosomes are 30–150 nm extracellular vesicles that play crucial roles in intercellular communication and hold significant potential as biomarkers for non-invasive liquid biopsy. However, the current isolation methods have limitations including being time-consuming, producing low yields, and having high costs. This study presents a novel automated exosome isolation method using EGCG-modified magnetic beads (EGCG@T) optimized for diverse biofluids including plasma, serum, urine, and saliva. We systematically investigated the optimal EGCG:T-Fe<sub>3</sub>O<sub>4</sub> ratio (0.1:1), binding time, elution parameters, and extraction buffer composition for each biofluid type. The developed protocol was successfully integrated into an automated workflow using the Nextractor<sup>®</sup> NX-Junior platform, combining exosome isolation and protein extraction into a single step. Western blot and ELISA analyses confirmed that the EGCG@T method yielded a significantly higher recovery of exosomal markers (CD9, CD63, CD81, TSG101, and ALIX) compared to conventional PEG precipitation, with the efficiency varying depending on the biofluid. Notably, CD63-positive exosomes were isolated with approximately two-fold higher efficiency from urine and 1.3-fold higher efficiency from saliva using the EGCG@T method. Our findings demonstrated that biofluid-specific optimization is essential for effective exosome isolation, as exosome subpopulations exhibited distinct physicochemical properties across different sample types. This automated, rapid, and efficient exosome isolation method provides a valuable platform for future clinical applications in non-invasive disease diagnosis and monitoring through liquid biopsy. |
| format | Article |
| id | doaj-art-020d91d9d58c40cf9c8626d005528b70 |
| institution | DOAJ |
| issn | 2076-3417 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
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| spelling | doaj-art-020d91d9d58c40cf9c8626d005528b702025-08-20T03:11:18ZengMDPI AGApplied Sciences2076-34172025-05-011511617010.3390/app15116170Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse BiofluidsHeejong Shin0Eunju Jeong1Seunggwan Lee2Department of Clinical Laboratory Sciences, College of Health Science, Korea University, Seoul 02841, Republic of KoreaGenolution Inc., Incheon 21999, Republic of KoreaDepartment of Clinical Laboratory Sciences, College of Health Science, Korea University, Seoul 02841, Republic of KoreaExosomes are 30–150 nm extracellular vesicles that play crucial roles in intercellular communication and hold significant potential as biomarkers for non-invasive liquid biopsy. However, the current isolation methods have limitations including being time-consuming, producing low yields, and having high costs. This study presents a novel automated exosome isolation method using EGCG-modified magnetic beads (EGCG@T) optimized for diverse biofluids including plasma, serum, urine, and saliva. We systematically investigated the optimal EGCG:T-Fe<sub>3</sub>O<sub>4</sub> ratio (0.1:1), binding time, elution parameters, and extraction buffer composition for each biofluid type. The developed protocol was successfully integrated into an automated workflow using the Nextractor<sup>®</sup> NX-Junior platform, combining exosome isolation and protein extraction into a single step. Western blot and ELISA analyses confirmed that the EGCG@T method yielded a significantly higher recovery of exosomal markers (CD9, CD63, CD81, TSG101, and ALIX) compared to conventional PEG precipitation, with the efficiency varying depending on the biofluid. Notably, CD63-positive exosomes were isolated with approximately two-fold higher efficiency from urine and 1.3-fold higher efficiency from saliva using the EGCG@T method. Our findings demonstrated that biofluid-specific optimization is essential for effective exosome isolation, as exosome subpopulations exhibited distinct physicochemical properties across different sample types. This automated, rapid, and efficient exosome isolation method provides a valuable platform for future clinical applications in non-invasive disease diagnosis and monitoring through liquid biopsy.https://www.mdpi.com/2076-3417/15/11/6170exosome isolationfunctionalized magnetic beadsepigallocatechin gallateautomated platformdiverse biofluids |
| spellingShingle | Heejong Shin Eunju Jeong Seunggwan Lee Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids Applied Sciences exosome isolation functionalized magnetic beads epigallocatechin gallate automated platform diverse biofluids |
| title | Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids |
| title_full | Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids |
| title_fullStr | Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids |
| title_full_unstemmed | Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids |
| title_short | Development of Automated Exosome Isolation Method Using Epigallocatechin Gallate-Modified Magnetic Beads: Standardized Protocols for Diverse Biofluids |
| title_sort | development of automated exosome isolation method using epigallocatechin gallate modified magnetic beads standardized protocols for diverse biofluids |
| topic | exosome isolation functionalized magnetic beads epigallocatechin gallate automated platform diverse biofluids |
| url | https://www.mdpi.com/2076-3417/15/11/6170 |
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