Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy

Pepijn Beekman, Agustin Enciso-Martinez, Hoon Suk Rho, Sidharam Pundlik Pujari, Aufried Lenferink, Han Zuilhof, Leon W.M.M. Terstappen, Cees Otto*, Séverine Le Gac

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Scopus)

Abstract

Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 μm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.

Original languageEnglish
Pages (from-to)2526-2536
Number of pages11
JournalLab on a Chip
Volume19
Issue number15
DOIs
Publication statusPublished - 11 Jul 2019

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Raman Spectrum Analysis
Raman spectroscopy
Tumors
Stainless Steel
Scanning electron microscopy
Stainless steel
Neoplasms
Antibodies
Cyanoacrylates
Substrates
Glues
Lab-On-A-Chip Devices
Modal analysis
Adhesives
Navigation
Blood
Imaging techniques
Cell adhesion
Cell Adhesion Molecules
Dermatoglyphics

Cite this

Beekman, Pepijn ; Enciso-Martinez, Agustin ; Rho, Hoon Suk ; Pujari, Sidharam Pundlik ; Lenferink, Aufried ; Zuilhof, Han ; Terstappen, Leon W.M.M. ; Otto, Cees ; Le Gac, Séverine. / Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy. In: Lab on a Chip. 2019 ; Vol. 19, No. 15. pp. 2526-2536.
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abstract = "Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 μm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.",
author = "Pepijn Beekman and Agustin Enciso-Martinez and Rho, {Hoon Suk} and Pujari, {Sidharam Pundlik} and Aufried Lenferink and Han Zuilhof and Terstappen, {Leon W.M.M.} and Cees Otto and {Le Gac}, S{\'e}verine",
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Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy. / Beekman, Pepijn; Enciso-Martinez, Agustin; Rho, Hoon Suk; Pujari, Sidharam Pundlik; Lenferink, Aufried; Zuilhof, Han; Terstappen, Leon W.M.M.; Otto, Cees; Le Gac, Séverine.

In: Lab on a Chip, Vol. 19, No. 15, 11.07.2019, p. 2526-2536.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy

AU - Beekman, Pepijn

AU - Enciso-Martinez, Agustin

AU - Rho, Hoon Suk

AU - Pujari, Sidharam Pundlik

AU - Lenferink, Aufried

AU - Zuilhof, Han

AU - Terstappen, Leon W.M.M.

AU - Otto, Cees

AU - Le Gac, Séverine

PY - 2019/7/11

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N2 - Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 μm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.

AB - Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 μm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.

U2 - 10.1039/c9lc00081j

DO - 10.1039/c9lc00081j

M3 - Article

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SP - 2526

EP - 2536

JO - Lab on a Chip

JF - Lab on a Chip

SN - 1473-0197

IS - 15

ER -