IMMUNOCAPTURING OF EXTRACELLULAR VESICLES ON STAINLESS
STEEL FOR MULTI-MODAL INDIVIDUAL CHARACTERIZATION WITH
CORRELATIVE LIGHT, ELECTRON AND PROBE MICROSCOPY
Pepijn Beekman
1,2,3, Agustin Enciso Martinez
2, Leon Terstappen
2, Cees Otto
2, Séverine Le Gac
11
Applied Microfluidics for BioEngineering Research, University of Twente, THE NETHERLANDS
2Medical Cell BioPhysics, University of Twente, THE NETHERLANDS
3
Laboratory of Organic Chemistry, Wageningen University & Research, THE NETHERLANDS
ABSTRACT
Here, we report a robust platform for multi-modal analysis of immuno-captured individual extracellular vesicles (EVs). Stainless steel substrates were surface-modified to covalently immobilize specific antibodies targeting proteins found on EVs. Using PDMS microchannels, EVs were selectively captured on the substrates. Next, individual EVs were retraced and correlatively characterized here using SEM, AFM and Raman Spectroscopy.
KEYWORDS: Extracellular vesicles, AFM, SEM, Raman, Correlative light, electron and probe microscopy INTRODUCTION
EVs are membrane-bound carriers of biomolecules in the size range of 30 nm – 1 μm that have been identified as potential biomarkers for real-time cancer patient management, as an alternative to circulating tumor cells (CTCs) [1]. To reliably quantify EVs in blood or other bodily fluids, more information about their morphology and chemical composition is needed. For that purpose, a large dataset of pure, single EVs of known origin, is required to derive a spectral fingerprint unique to ominous EVs. For a reliable study of EVs, correlating data acquired with different analytical techniques allows unambiguously confirming the nature of the object being analyzed. Raman Spectroscopy (RS) provides chemical information on the EVs. SEM gives a quick validation of the capturing and size and coverage of the EVs. AFM grants high resolution, quasi-3D morphology information. To that end, here we developed a platform compatible with all these analytical techniques, for the immunocapture of EVs (Fig. 1).
EXPERIMENTAL
Polished stainless steel (SS) was identified as a suitable material for this multi-modal analysis, since it is smooth, conductive and gives low background signal in RS. Stainless steel substrates were chemically modified with a carboxydecyl phosphonic acid (CDPA) monolayer [2], onto which anti-EpCAM antibodies were covalently attached. The resulting monolayers were characterized by IR spectroscopy and XPS. EVs obtained from a human prostate cancer cell line (LNCaP) were captured on the substrate using a PDMS microfluidic channel (0.2 mm height and 4 mm width).
RESULTS AND DISCUSSION
XPS and IR spectroscopy reveal the formation of a dense and well-ordered CDPA monolayer. Antibody immobilization was confirmed using fluorescence microscopy. EVs were successfully captured on modified SS substrates with good coverage (Fig. 2), whereas no EV was detected in three different negative control experiments (no activation of the carboxyl group, no antibody or no EV sample). Direct evidence
Figure 1: Individual, selectively captured
___EV were characterized by AFM, SEM and
___Raman.
of the presence of EVs was given by Raman spectroscopy, which shows a clear lipid band at 2851 cm-1 (CH 2
symmetric stretch of lipids) (Fig. 3). Raman spectra as shown in Fig. 3 were obtained after hierarchical cluster analysis (HCA) which was performed based on principal component analysis (PCA) scores. Uncured PDMS oligo-mers were initially identified in the EV membranes (Fig. 3, red). In other samples using more thoroughly cured microchannels, no PDMS was found in the EVs (Fig. 3, green). Using AFM, many more smaller objects were detected than in SEM. A density of 4.3x105 particles.mm-2 was found, with an average diameter of 101±111 nm
978-0-578-40530-8/µTAS 2018/$20©18CBMS-0001 2402 22nd International Conference on Miniaturized
Systems for Chemistry and Life Sciences November 11-15, 2018, Kaohsiung, Taiwan
(n=5), corresponding to a capturing efficiency of 38% (based on Nanoparticle Tracking Analysis (NTA) suggesting a concentration of 1.06x108 EV ml-1). The size distribution is presented in Fig. 4, and compared to that obtained
from NTA in suspension.
Figure 2: The same region imaged with multiple analytical instruments. a) SEM image recorded (in absence of gold coating) at 5 kV. b) Raman cluster image shows a 2-level Raman map (scanning step size: 470 nm) resulting from hierarchical cluster analysis (HCA) based on PCA scores. c) AFM amplitude map. d) Composite image of a), b) and c).
Figure 3: Normalized Raman spectra of EVs (Fig. 2b, red pixels), background (Fig. 2b, black pixels), and EV without PDMS measured in a different sample (green). EV spectra show a characteristic lipid Raman band at 2851 cm-1.
Figure 4: Size distribution of surface-immobilized EVs as de-termined by AFM after capture on the surface (red), corre-sponding to NTA data obtained on EVs in suspension (blue). Error bars show the standard deviation (n=5).
CONCLUSION
The herein reported methodology opens avenues to combine immunocapturing with multi-modal analysis using various scanning probe, light and electron microscopy techniques to study biological microparticles of interest.
ACKNOWLEDGEMENTS
We thank prof. Han Zuilhof for his assistance with the surface modification and dr. Hoon Suk Rho for the design and fabrication of the PDMS molds. This project is funded by Netherlands Organisation for Scientific Re-search - Domain Applied and Engineering Sciences (NWO-TTW).
REFERENCES
[1] Coumans F.A. et al., “All circulating EpCAM+CK+ CD45- objects predict overall survival in
castration-resistant prostate cancer”, Annals of Oncology 21: 1851–1857, 2010
[2] Kosian, M. et al., “Structure and Long-Term Stability of Alkylphosphonic Acid Monolayers on SS316L
Stain-less Steel, Langmuir”, 32 (4), pp 1047–1057, 2016
CONTACT
* S. Le Gac; phone: +31-53-489-2722; s.legac@utwente.nl
2700 2800 2900 3000 3100 3200 20 40 60 80 100 Raman shift [cm-1 ] Offset intens ity values (ar b. units) Background EVs + PDMS EVs 2851 2403
