Cargando…
Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo
The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving ce...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Association for the Advancement of Science
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413728/ https://www.ncbi.nlm.nih.gov/pubmed/32821836 http://dx.doi.org/10.1126/sciadv.abb3521 |
_version_ | 1783568850776752128 |
---|---|
author | Wang, Xixian Xin, Yi Ren, Lihui Sun, Zheng Zhu, Pengfei Ji, Yuetong Li, Chunyu Xu, Jian Ma, Bo |
author_facet | Wang, Xixian Xin, Yi Ren, Lihui Sun, Zheng Zhu, Pengfei Ji, Yuetong Li, Chunyu Xu, Jian Ma, Bo |
author_sort | Wang, Xixian |
collection | PubMed |
description | The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min(−1) throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min(−1). From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application. |
format | Online Article Text |
id | pubmed-7413728 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American Association for the Advancement of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-74137282020-08-19 Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo Wang, Xixian Xin, Yi Ren, Lihui Sun, Zheng Zhu, Pengfei Ji, Yuetong Li, Chunyu Xu, Jian Ma, Bo Sci Adv Research Articles The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min(−1) throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min(−1). From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application. American Association for the Advancement of Science 2020-08-07 /pmc/articles/PMC7413728/ /pubmed/32821836 http://dx.doi.org/10.1126/sciadv.abb3521 Text en Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (https://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. |
spellingShingle | Research Articles Wang, Xixian Xin, Yi Ren, Lihui Sun, Zheng Zhu, Pengfei Ji, Yuetong Li, Chunyu Xu, Jian Ma, Bo Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
title | Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
title_full | Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
title_fullStr | Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
title_full_unstemmed | Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
title_short | Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
title_sort | positive dielectrophoresis–based raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413728/ https://www.ncbi.nlm.nih.gov/pubmed/32821836 http://dx.doi.org/10.1126/sciadv.abb3521 |
work_keys_str_mv | AT wangxixian positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT xinyi positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT renlihui positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT sunzheng positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT zhupengfei positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT jiyuetong positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT lichunyu positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT xujian positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo AT mabo positivedielectrophoresisbasedramanactivateddropletsortingforculturefreeandlabelfreescreeningofenzymefunctioninvivo |