Cargando…
A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles
The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831566/ https://www.ncbi.nlm.nih.gov/pubmed/31690751 http://dx.doi.org/10.1038/s41598-019-52366-4 |
_version_ | 1783465998641266688 |
---|---|
author | Brittain, George C. Chen, Yong Q. Martinez, Edgar Tang, Vera A. Renner, Tyler M. Langlois, Marc-André Gulnik, Sergei |
author_facet | Brittain, George C. Chen, Yong Q. Martinez, Edgar Tang, Vera A. Renner, Tyler M. Langlois, Marc-André Gulnik, Sergei |
author_sort | Brittain, George C. |
collection | PubMed |
description | The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages. |
format | Online Article Text |
id | pubmed-6831566 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-68315662019-11-13 A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles Brittain, George C. Chen, Yong Q. Martinez, Edgar Tang, Vera A. Renner, Tyler M. Langlois, Marc-André Gulnik, Sergei Sci Rep Article The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages. Nature Publishing Group UK 2019-11-05 /pmc/articles/PMC6831566/ /pubmed/31690751 http://dx.doi.org/10.1038/s41598-019-52366-4 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Brittain, George C. Chen, Yong Q. Martinez, Edgar Tang, Vera A. Renner, Tyler M. Langlois, Marc-André Gulnik, Sergei A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles |
title | A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles |
title_full | A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles |
title_fullStr | A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles |
title_full_unstemmed | A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles |
title_short | A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles |
title_sort | novel semiconductor-based flow cytometer with enhanced light-scatter sensitivity for the analysis of biological nanoparticles |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831566/ https://www.ncbi.nlm.nih.gov/pubmed/31690751 http://dx.doi.org/10.1038/s41598-019-52366-4 |
work_keys_str_mv | AT brittaingeorgec anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT chenyongq anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT martinezedgar anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT tangveraa anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT rennertylerm anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT langloismarcandre anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT gulniksergei anovelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT brittaingeorgec novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT chenyongq novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT martinezedgar novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT tangveraa novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT rennertylerm novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT langloismarcandre novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles AT gulniksergei novelsemiconductorbasedflowcytometerwithenhancedlightscattersensitivityfortheanalysisofbiologicalnanoparticles |