Microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes

Abstract

Exosomes are nanoscale vesicles found in many bodily fluids which play a significant role in cell-to-cell signaling and contain biomolecules indicative of their cells of origin. Recently, microfluidic devices have provided the ability to efficiently capture exosomes based on specific membrane biomarkers, but releasing the captured exosomes intact and label-free for downstream characterization and experimentation remains a challenge. We present a herringbone-grooved microfluidic device which is covalently functionalized with antibodies against general and cancer exosome membrane biomarkers (CD9 and EpCAM) to isolate exosomes from small volumes of high-grade serous ovarian cancer (HGSOC) serum. Following capture, intact exosomes are released label-free using a low pH buffer and immediately neutralized downstream to ensure their stability. Characterization of captured and released exosomes was performed using fluorescence microscopy, nanoparticle tracking analysis, flow-cytometry, and SEM. Our results demonstrate the successful isolation of intact and label-free exosomes, indicate that the amount of both total and EpCAM+ exosomes increases with HGSOC disease progression, and demonstrate the downstream internalization of isolated exosomes by OVCAR8 cells. This device and approach can be utilized for a nearly limitless range of downstream exosome analytical and experimental techniques, both on and off-chip.

A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum

“…Despite this, the potential use of other RNA and protein targets identified in EVs secreted by cancer cell lines in vitro is hindered by challenges in biomarker validation in patient samples. Although affinity capture of cancer specific EVs from patient serum has shown some success in ovarian cancer 58 , there is currently no standardised method for isolating and purifying circulating EVs from human serum.…”

Towards establishing Extracellular Vesicle-associated RNAs as biomarkers for HER2+ breast cancer

“…EpCAM is a cell adhesion glycoprotein which has been used extensively as a liquid biopsy marker for several epithelial cancers [15], whilst HER2 plays an important role in breast cancer subtyping.…”

Progress in Microfluidics‐Based Exosome Separation and Detection Technologies for Diagnostic Applications

“…Because of these advantages, microfluidics has been successfully applied in the biomedical field, for example, in particle separation, cell separation, DNA/RNA manipulation, polymerase chain reaction (PCR), detection of circulating tumor cells (CTCs) and droplet generation . In recent years, increasing attention has been paid to the development of new exosome separation and detection techniques based on microfluidics . These new techniques have exhibited high potential application prospects because of their simple, fast, and time‐saving procedures, as well as the minimal consumption of samples and reagents.…”