Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Abstract

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform

“…Microfluidic designs presented here could be readily extended by increasing the number of independent channels common to more complex microfluidic devices 61 , with the size and complexity of the device becoming the ultimate limits in terms of multiplexing. Nevertheless, as a complimentary route, our platform is also fully compatible with single-molecule fluorescence read-out approaches 62 , and thus could be combined with state-of-the-art fluorescently-tagged antibody 3 , 4 , 63 , 64 or aptamer 58 , 65 libraries to enable large-scale single particle profiling. We envision that our platform, when combined with on-chip standard additions approaches 66 and improved surface passivation could enable diagnostic and care monitoring of diseases based on a selection of disease biomarkers 60 .…”

Engineering Cell Membranes: From Extraction Strategies to Emerging Biosensing Applications

“…Therefore, research on protein sensing based on extracted cell membranes is in full swing. 110 As an essential category of biomarkers, membrane proteins are responsible for signaling between a cell and its external environment, moving ions/molecules across the membrane and allowing it to recognize other cells as well as attach to the surface. 111 For example, anti-CD44 Ab (Ab1) was used to be modified on the nanoparticles interface to block the nonspecific adsorption and capture the CD44 selectively.…”

“…Proteins are commonly identified as biomarkers for tracking or identifying various diseases in organisms. , Delineating and identifying the spatial distribution of human proteins at the organ, tissue, and cellular level can offer insight into health and disease and represent an excellent reference for the discovery of biomarkers, therapeutic targets, and disease signatures. − In this regard, the abundant composition of cellular active species on the surface of extracted cell membranes confers extraordinary protein recognition capabilities, making them widely adopted for the specific detection of a multitude of protein-based disease markers. Therefore, research on protein sensing based on extracted cell membranes is in full swing …”

Stoichiometric constraints for detection of EV-borne biomarkers in blood

“…The attempts to extend the number of detected EV displayed proteins are still in domain of research and development, most remarkably including single EV immuno-sequencing [146]. Although both microarrays (custom antibody spotted chips [187]) and bead-based arrays [188] have been used to-date for multiplex protein detection on intact EVs, obtained results for distinct markers, or combinations thereof, are averaged across the whole EV population, while the individual EV profiles remain limited to a very few proteins. The combination of fluorescence-based target detection with optically encoded beads such as those provided by Luminex, Miltenyi (MACSPlex), or Quanterix (SiMoA) can increase the multiplex capacity.…”