Multiplexed Profiling of Extracellular Vesicles for Biomarker Development

Abstract

Extracellular vesicles (EVs) are cell-derived membranous particles that play a crucial role in molecular trafficking, intercellular transport and the egress of unwanted proteins. They have been implicated in many diseases including cancer and neurodegeneration. EVs are detected in all bodily fluids, and their protein and nucleic acid content offers a means of assessing the status of the cells from which they originated. As such, they provide opportunities in biomarker discovery for diagnosis, prognosis or the stratification of diseases as well as an objective monitoring of therapies. The simultaneous assaying of multiple EV-derived markers will be required for an impactful practical application, and multiplexing platforms have evolved with the potential to achieve this. Herein, we provide a comprehensive overview of the currently available multiplexing platforms for EV analysis, with a primary focus on miniaturized and integrated devices that offer potential step changes in analytical power, throughput and consistency.

Keywords: Biomarker; Exosomes; Extracellular vesicles; Liquid biopsy; Multiplexed profiling; Point-of-care.

Fig. 1

Potential clinical applications of composite EV biomarkers. a The molecular profiling of EVs may be based on proteomics (e.g. membrane proteins and internal proteins), RNAs or metabolites (e.g. lipids and glycans). b The multiplexed analysis of EV components can generate a box plot representation of expression levels in A and B groups as defined in c. c Potential clinical applications of EV markers

Fig. 2

Main external-coding strategies for EV profiling. Multiplexing is typically based on the combination of multiple receptors with one of the four coding strategies. Depending on how the analyte signal is generated and transduced, multiple external codes generated by chemical reporter labelling, physical spatial coding, biological coding, or nanoparticle coding, can be used in association with multiple receptors (QDs = quantum dots, NP = nanoparticles)

Fig. 3

Typical SERS-based approaches for EV multiplexing. a An examination of multiple EV components using the bulk chemical fingerprints of immobilized EVs. Adapted with permission from Ref. [35]. Copyright 2018 American Chemical Society. b Schematic illustration of molecular phenotype profiling of CD63-positive EVs using SERS nanotags (antibody-Raman dye conjugate: anti-MIL38-DTNB, anti-EpCAM-MBA, and anti-CD44V6-TFMBA). Adapted with permission from Ref. [36]. Copyright 2020 American Chemical Society. TFMBA: 2,3,5,6-Tetrafluoro-4-mercaptobenzonic acid, DTNB: 5,5’-dithiobis(2-nitrobenzoic acid), MBA: 4-mercaptobenzoic acid. c A multiplex EV phenotype assay chip using four SERS nanotags. The phenotypic evolution can be tracked by analysing EV samples before, during, and after immunotherapy treatment, thus providing information on treatment responses and the early signs of drug resistance. Adapted with permission from Ref. [39]. Copyright 2020 American Association for the Advancement of Science (AAAS)

Fig. 4

Multiplexed profiling of EV proteins using fluorescent dye-based chemical coding strategy. a The ExoSearch chip for continuous mixing, isolation and in situ, multiplexed detection of circulating exosomal markers CA-125, EpCAM and CD24. Reproduced with permission from Ref. [43]. Copyright 2016 Royal Society of Chemistry. b Multiplexed single-EV analysis by microfluidic immunofluorescence staining. Reproduced with permission from Ref. [51]. Copyright 2018 American Chemical Society. c The principle of an enzyme-aided fluorescence amplification based on GO-aptamer interactions for the detection of exosomal membrane proteins. Reproduced with permission from Ref. [53]. Copyright 2018 Elsevier

Fig. 5

Chemical coding strategies with signal amplification for EV multiplexing. a An aptasensor for the thermophoretic enrichment of EVs and multiplexed profiling of their surface proteins. Reproduced with permission from Ref. [57]. Copyright 2019 Springer Nature. b DNA ligation system for EV membrane protein profiling using thermophoresis. Reproduced with permission from Ref. [69]. Copyright 2021 American Chemical Society. c Schematic of the TPEX microfluidic multiplexing platform. Exosomes were incubated with different fluorescent aptamers, either individually (singleplex) or as a mixture (multiplex), for templated plasmonics for exosome (TPEX) analysis. Reproduced with permission from Ref. [63]. Copyright 2020 American Association for the Advancement of Science (AAAS). d Thermophoretic sensor implemented with nanoflares for in situ detection of exosomal miRNAs. Reproduced with permission from Ref. [70]. Copyright 2020 American Chemical Society

Fig. 6

a Molecular beacon-based exosome internal RNA triplexing (F: fluorescent dye. Q: quencher). Reproduced with permission from Ref. [74]. Copyright 2016 Elsevier. b Simultaneous in situ detection of EV membrane protein and internal miRNA using dye conjugated molecular beacons and dye conjugated antibodies, respectively. Reproduced with permission from Ref. [76]. Copyright 2021 MDPI. c Simultaneous in situ detection of exosomal protein markers (CD81, ephrin type-A receptor 2, carbohydrate antigen 19–9) and miRNAs (miRNA-451a, miRNA-21, miRNA-10b) using QDs labelled antibody and molecular beacons using fusogenic vesicles in a microfluidic device. Reproduced with permission from Ref. [77]. Copyright 2020 John Wiley & Sons

Fig.7

Schematic view of a physical spatial coding-based SPR platform for EV multiplexing. a Antibodies specific to EV transmembrane proteins were printed on the gilded gold chip, and integrated into a flow cell. Reproduced with permission from Ref. [96]. Copyright 2014 American Chemical Society. b nPLEX chip. (i) Integration of a multi-channel microfluidic cell for independent and parallel analyses. Transmission intensities of 12 × 3 nanohole arrays were measured simultaneously using the imaging setup. (ii) A representative schematic of changes in transmission spectra showing EV detection with nPLEX. (iii) Ascites-derived exosomes from ovarian cancer and noncancer patients were evaluated by the nPLEX sensor. Cancer EVs were captured on EpCAM and CD24-specific sensor sites with associated intensity changes in the transmitted light. Adapted with permission from Ref. [86]. Copyright 2014 Springer Nature. c Enzymatic amplified plasmonic sensor for EV multiplexing. Reproduced with permission from Ref. [117]. Copyright 2019 Springer Nature. d Surface plasmon-enhanced fluorescence biosensing for EV multiplexed profiling. Reproduced with permission from Ref. [118]. Copyright 2020 John Wiley and Sons. e Intravesicular nanoplasmonic system for EV multiplexing. Reproduced with permission from Ref. [94]. Copyright 2018 American Chemical Society

Fig. 8

Non-SPR physical coding-based multiplexed profiling of EVs. a Schematic view of EV array detection of EV proteins. Adapted with permission from Ref. [123]. Copyright 2013 Taylor and Francis. b Integrated magnetic–electrochemical exosome (iMEX) platform. The sensor can simultaneously measure signals from eight parallel electrodes. Reproduced with permission from Ref. [92]. Copyright 2016 American Chemical Society. c Antibody modified cantilevers in the array with a reference control for differential detection of signal (up). Schematic of the effect of the nanoparticle mass loading on the nanomechanical deflection of the cantilever (down). Adapted with permission from Ref. [140]. Copyright 2016 Royal Society of Chemistry. d Schematic representation of the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) detection process. SP-IRIS detection principle, monochromatic LED light illuminates the sensor surface and the interferometrically enhanced nanoparticle scattering signature is captured on a CMOS camera (left). Low-magnification interferometric image showing microarray of immobilized capture probes (right). Reproduced with permission from Ref. [141]. Copyright 2016 Springer Nature. e Schematic diagram of an integrated microfluidic chip for plasma separation, EV detection, and molecular analysis. Reproduced with permission from Ref. [100]. Copyright 2020 American Chemical Society. f Multi-test line strip for profiling of EV membrane proteins. Reproduced with permission from Ref. [142]. Copyright 2017 Elsevier

Fig. 9

a Schematic assay format of immuno-PCR assisted multiplex detection of membrane proteins on EVs using capillary electrophoresis. The standard curve of the peak area in an electropherogram vs number of exosomes per well for multiplex immuno-PCR (upper inset). The immuno-PCR peaks for the detection of CD9, CD34, CD117, CD123, and CD135 molecules (bottom inset). Adapted with permission from Ref. [155]. Copyright 2020 American Chemical Society. b The convergence of antibody-DNA labelling and digital PCR for EV multiplexing. Reproduced with permission from Ref. [157]. Copyright 2020 John Wiley and Sons

Fig. 10

Nanoparticle coding strategy for EV multiplexing using a fluorophore doped beads. Reproduced with permission from Ref. [165]. Copyright 2020 AAN Publications. b Quantum dots. Reproduced with permission from Ref. [185]. Copyright 2019 Springer. c Plasmonic nanoparticles. Reproduced with permission from Ref. [177]. Copyright 2017 Springer Nature. d Redox active Cu and Ag nanoparticles. Reproduced with permission [179]. Copyright 2014 John Wiley & Sons

Fig. 11

Flowchart of EV biomarker development powered by multiplexing platform