Digital Detection of Exosomes by Interferometric Imaging.

Chiara Piotto,Davide Prosperi,Marcella Chiari,Ayca Yalcin Ozkumur,George G Daaboul,Paolo Bettotti,Luisa Benussi,Benedetta Santini,David S Freedman,Marina Cretich,Paola Gagni,Roberta Ghidoni,M Selim Ünlü,Miriam Ciani

doi:10.1038/srep37246

Abstract

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.

Highlights

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics
In this work we demonstrate the use of an innovative technological platform termed Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) for the high-throughput characterization of exosome concentration, relative size distribution and phenotyping in biological fluids[28]
The detection principle for SP-IRIS is based on the enhanced contrast in the scattering signal from particles captured on a silicon substrate with a thin silicon dioxide layer (Fig. 1)

Summary

Introduction

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. In order to overcome the limitations of conventional NTA to determine the cell of origin and to distinguish between different vesicles types (i.e. EVs, lipids and protein aggregates), a short wavelength (405-nm blue-violet) laser and a high sensitivity camera to detect fluorescent particles[19] are incorporated to the optical system In this modality, combining NTA with fluorescence measurements, only exosomes labeled with specific fluorescent antibodies are detected, allowing their phenotype to be determined. A real-time, label-free sensing of single exosomes in serum using antibody functionalized micro-toroid optical resonators has been introduced[21] Highly sensitive, such high-Q optical resonators have significant challenges of identifying size of captured particles in a complex solution and difficulty of multiplexing

Methods

Results

Conclusion