EBS2019 Florence, Italy, February 18-21, 2019 Microarray based - - PowerPoint PPT Presentation

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EBS2019 Florence, Italy, February 18-21, 2019 Microarray based - - PowerPoint PPT Presentation

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2 European Biosensor Symposium 2019 EBS2019 Florence, Italy, February 18-21, 2019 Microarray based platform for extracellular vesicles imaging and phenotyping D. Brambilla, M. Cretich, A. Romanato, M. Odinolfi, E. Chiodi, S. Descroix, A.

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2° European Biosensor Symposium 2019

EBS2019

Florence, Italy, February 18-21, 2019

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Microarray based platform for extracellular vesicles imaging and phenotyping

  • D. Brambilla, M. Cretich, A. Romanato, M. Odinolfi, E. Chiodi,
  • S. Descroix, A. Yalcin Ozkumur, M.S. Unlu, M. Chiari
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ü Microarray platform for Exosome Immune-Capture üInterferometric detection of Single NanoParticles

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“Extracellular vesicles: Exosomes, microvesicles, and friends” G. Raposo and W. Stoorvogel - J. Cell Biol. Vol. 200 No. 4 373–383

Extracellular Vesicles

Exosomes

q Early Endosome q Multi Vesicular Bodies q Lysosomial degradation q Exosome Release

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Exosome: Diagnostic potential

q Mirrors the cell of origin q Liquid Biopsy ü Cancer ü Neurodegenerative diseases

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Exosome: Characteristics

ü Number ü Size distribution ü Membrane composition ü Cargo

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Exosome: Capture

ü Microarray platform for Exosome Immune-Capture

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Microarray technique

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Polymeric Coating

Copoly(DMA-NAS-MAPS)

m=97 n=2 p=1

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Probe Immobilization

Antibodies (proteic probes) ü Tetraspanins (CD9, CD63)

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DNA-directed Immobilization #1

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DNA-directed Immobilization #2

ü DNA Spot morphology ü Antibody specificity ü Improved flexibility

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Selectivity

ü Multiplexing

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Detection

ü Interferometric detection

  • f Single NanoParticles
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SP-IRIS: Sizing and Phenotyping #1

ü IRIS: Interferometric Reflectance Imaging System § Led-based technique § Matter immobilized on surface § Shifts of reflectivity Image contrast

  • G. G. Daaboul et al. (2011) “LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular

interactions” Biosensors and Bioelectronics, 26, 5, 2221-2227.

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SP-IRIS: Sizing and Phenotyping #2

  • G. G. Daaboul et al. (2016) “Digital Detection of Exosomes by Interferometric Imaging” Scientific Reports, 6:37246.
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  • G. G. Daaboul et al. (2016) “Digital Detection of Exosomes by Interferometric Imaging” Scientific Reports, 6:37246.

ü Digital count of nanoparticles (50-200 nm) ü Size distribution ü Phenotyping (microarray platform)

SP-IRIS: Sizing and Phenotyping #3

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Exosome Immune-capture: Results #1

Spot: DNA-directed antiCD9 Incubation: Human Blood Serum, 2.5h, RT

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Exosome Immune-capture: Results #2

Spot: DNA-directed antiCD9 Incubation: Human Blood Serum, 2.5h, RT

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Exosome Immune-capture: Results #3

Spot: DNA-directed antiCD9 Incubation: Human Blood Serum, 2.5h, RT

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Exosome Immune-capture: Results #4

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Exosome Immune-capture: Results #5

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Exosome Immune-capture: Results #6

unmodified AntiCD63 DNA-Directed AntiCD63

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Next Step: Real Time SP-IRIS

ü Kd constant Affinity ü Distinguish specific and non-specific events

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Acknowledgments

FET-Open 2016-2017 Grant Agreement no. 766466

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THANK YOU FOR THE KIND ATTENTION