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Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets
In This Article
Summary
This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.
Abstract
Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential
Introduction
The growing interest in EV research in diagnosis and in therapeutics1,2,3,4,5, combined with the challenges this field faces, has resulted in the development and implementation of a large variety of approaches and techniques for quantifying or characterizing these vesicles. The most widely used methods for EV identification are protein-specific immunoblotting and proteomics to confirm the origin of EVs, transmission electron microscopy (TEM) to confirm their structure, and nanoparticle tracking analysis (NT....
Protocol
1. Gold substrate preparation
NOTE: Three types of surfaces are produced on gold chips: 1) naked surface, 2) chemically functionalized, and 3) biofunctionalized (ligands grafted on C11C16 layer). They will be called "naked," "C11C16," and "ligands," respectively, from this point onward.
- Gold substrate preparation:
NOTE: For this protocol, the gold biochips were manufactured in-house i.......
Representative Results
Determination of the optimum pH conditions for ligand grafting
The different ligands used to prepare the biochips are tested as a function of the pH and their availability to interact with the thiolate chemical layer (Figure 3). The ligands are diluted in acetate buffer at different pH values and injected on the biochip chemically functionalized with a C11C16 layer. The solutions are injected randomly on the surface, and a detergent (OG at 40 mM) is injected after each.......
Discussion
The recent methods for EV identification that are the most widely used are protein-specific immunoblotting to confirm the origin of EVs, TEM to confirm their structure, and NTA to quantify their number and size distribution in a sample volume3. Nevertheless, the high interest in EVs in (bio)medical research and the limitations of existing analytical tools have prompted the scientific community to develop new methods for EV characterization, discrimination, and quantification.
Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgements
Kelly Aubertin and Fabien Picot from the IVETh core facility (Paris) are acknowledged for the Raman imaging experiments. Thierry Burnouf (Taipei Medical University, Taiwan) and Zuzana Krupova (From Helincourt, France) are acknowledged for providing the EV samples derived from blood platelet and bovine milk samples, respectively. The work was supported by the region Bourgogne Franche-Comté and the EUR EIPHI graduate school (NOVICE project, 2021-2024). Part of this work was done using the CLIPP platform and in RENATECH clean room facilities in FEMTO-ENGINEERING, for which we thank Rabah Zeggari.
....Materials
| Name | Company | Catalog Number | Comments |
| CD41a antibody | Diaclone SAS (France) | 447528 | |
| CP920 | Microparticles GmbH, Germany | 448303 | |
| DXR3xi | Thermo Fisher Scientific | T1502 | |
| EDC | Sigma | A6272 | |
| Ethanolamine | Sigma | P5368-10PAK | |
| Evs derived from platelet concentrates | Collaboration : Pr T. Burnouf (TMU, Taipei) | S2889 | |
| Evs from bovine milk | Collaboration : Dr Z. Krupova (Excilone, Helincourt - France) | 3450 | |
| Glutaraldehyde | Sigma | 56845 | |
| Gwyddion | 853.223.020 | ||
| Magnetron sputtering | PLASSYS | SAB5300165 | |
| mercapto-1-hexadecanoic acid | Sigma | G5882 | |
| Mercapto-1-undecanol | Sigma | O8001 | |
| Mountains SPIP ones | Digital Surf | ||
| NanoWizard 3 Bioscience | Bruker-JPK | ||
| Octyl Glucoside (OG) | Sigma | ||
| Ovalbumine antibody | Sigma | ||
| Phosphate Buffer Saline (PBS) | Sigma | ||
| Rat Albumin Serum (RSA) | Sigma | ||
| Sodium acetate buffer | Sigma | ||
| SPR-Biacore 3000 | GE Healthcare/ Cytiva life sciences | ||
| SPRi Biochip | MIMENTO technology platform | The biochips were produced in-house in the clean room, Besancon | |
| SPRi Plex II | Horiba Scientific | ||
| Sulfo-NHS | Sigma |
References
- Silva, A. K. A., et al. Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France". Advanced Drug Delivery Reviews. 179, 114001 (2021).
- Xunian, Z., Kalluri, R.
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