EV-Blade: an automated centrifugal-pneumatic cartridge for size- and affinity-based exosome isolation from whole...

Extracellular vesicles (EVs), especially the exosome-sized subset are increasingly exploited as minimally invasive cancer biomarkers. These small vesicles are abundant in biofluids and play crucial roles in intercellular communication and disease progression by transporting bioactive molecules. Exosomes offer distinct diagnostic and prognostic advantages over traditional cancer biomarkers, but purifying and extracting exosomes from blood remains challenging. There is a need to simply and cost-effectively isolate exosomes from milliliter quantities of whole blood for transcriptional and other omics-based research. Addressing this gap, we propose a microfluidic cartridge, the EV-Blade, for size and affinity-based purification of exosomes on a centrifugal microfluidic platform. We demonstrate a method to automate exosome purification from whole blood samples on a single microfluidic cartridge. The EV-Blade system combines blood centrifugation, plasma filtration for EV size selection and immunomagnetic capture using functionalized superparamagnetic nanoparticles targeting CD9, CD63, and CD81 exosomal surface proteins. We report on the device performance, purity of exosome recovery and the quality of RNA collected following on-chip EV lysis. We use this automated method to detect relevant long coding and non-coding RNA transcripts in circulating blood exosomes, showcasing the EV-Blade for use in cancer patient risk stratification. The system presented herein represents a significant advancement in exosome purification, offering a robust and automated platform for liquid biopsy-based cancer research and clinical applications. This innovation holds promise for cancer diagnosis, prognosis, and monitoring through non-invasive biomarkers.