Description
Extracellular vesicles (EVs) are nanometre-sized double-layered phospholipid vesicles that circulate in body fluids, carrying genetic information from their parent cells, making them highly suitable for liquid biopsy[1]. Despite their value, their current use in clinical practice is still limited. Among the limiting factors, one of the most critical is their isolation. Conventional approaches are characterised by low purity and scarce throughput, or poor reproducibility[2]. Here, we propose a platform for EV isolation by affinity capture using magnetic beads, based on droplet microfluidics[3]. Monophasic microfluidic has been used for this scope with a low capture efficiency[4], mainly due to bead sedimentation and low throughput, being limited to a few tens of microlitres, whereas clinical applications require larger sample volumes (0.5 – 1 ml). On the contrary, by confining both samples and beads in droplets, the beads cannot escape the droplet itself, and importantly, the spontaneous formation of recirculation zones within the droplet promotes the mixing. In our case, beads are functionalised with Anti-CD9 antibodies targeting specific EV tetraspanins present on the membrane. The microfluidic device is fabricated by replica molding and being coupled with a series of low-cost optical sensors, syringe pumps and pressure controllers. The entire isolation process operates in a relatively short time (about 4.5 hours), processing three samples at a time and handling sample volumes of up to 2 ml per sample. The platform was initially validated from the microfluidic point of view: throughput, automation, magnetic bead handling[5]. The EV isolation capability from human plasma samples was then investigated among three different techniques using microfluidics, in-batch protocols, and ultracentrifugation, operating a systematic comparison with commonly used methods. Fresh human plasma from healthy donors was used, without the need for preconcentration steps. Isolated EVs were characterized by the commonly used techniques (NTA, confocal microscopy, BCA, and WB) showing very positive outcomes. In particular, we obtained about 60% EV isolation efficiency (by NTA and BCA) and positive WB analysis (CD9, CD29), showing a 10-fold higher signal from the samples processed by the microfluidic platform rather than the in-batch protocol. Moreover, the microfluidic samples result definitely purer in the target proteins than the ultracentrifuge samples. Dedicated experiments for EV elution and in-droplet analysis are under investigation.
Given these results, we strongly believe that droplet microfluidics represents a promising technology for the isolation in clinical practice.
[1] Raposo G. et al, Nat Rev Mol Cell Biol 20, 2019
[2] Momen-Heravi F. et al, Biol Chem 394, 2013
[3] Moragues T. et al, Nat Rev Methods Primers 3, 2023
[4] Zhao Z. et al, Lab Chip 16, 2016
[5] Meggiolaro A. et al, Sens. Actuators B Chem 409, 2024