Tumor-derived circulating exosomes, enriched with a group of tumor antigens, have been recognized as a promising biomarker source for cancer diagnosis via less invasive procedure. Quantitatively pinpointing exosome tumor markers is appealing, yet challenging. In this study, we developed a simple microfluidic approach (ExoSearch) which provides enriched preparation of blood plasma exosomes for in-situ, multiplexed detection using immunomagnetic beads. The ExosSearch chip offers robust, continuous-flow design for quantitative isolation and release of blood plasma exosomes in a wide range of preparation volumes (10 μL to 10 mL). We employed the ExoSearch chip for blood-based diagnosis of ovarian cancer by multiplexed measurement of three exosomal tumor markers (CA-125, EpCAM, CD24) using a training set of ovarian cancer patient plasma, which showed significant diagnostic power (a.u.c. = 1.0, p = 0.001) and was comparable with standard Bradford assay. This work provides an essentially needed platform for utilization of exosomes in clinical cancer diagnosis, as well as fundamental exosome research.

Longitudinal cancer monitoring is crucial to clinical implementation of precision medicine. There is growing evidence indicating important functions of extracellular vesicles (EVs) in tumor progression and metastasis, including matrix remodeling via transporting matrix metalloproteases (MMPs). However, the clinical relevance of EVs remains largely undetermined, partially owing to challenges in EV analysis. Distinct from existing technologies mostly focused on characterizing molecular constituents of EVs, here we report a nanoengineered lab-on-a-chip system that enables integrative functional and molecular phenotyping of tumor-associated EVs. A generalized, high-resolution colloidal inkjet printing method was developed to allow robust and scalable manufacturing of three-dimensional (3D) nanopatterned devices. With this nanochip platform, we demonstrated integrative analysis of the expression and proteolytic activity of MMP14 on EVs to detect in vitro cell invasiveness and monitor in vivo tumor metastasis, using cancer cell lines and mouse models. Analysis of clinical plasma specimen showed that our technology could be used for cancer detection including accurate classification of age-matched controls and patients with ductal carcinoma in situ, invasive ductal carcinoma, or locally metastatic breast cancer in a training cohort (n = 30, 96.7% accuracy) and an independent validation cohort (n = 70, 92.9% accuracy). With clinical validation, our technology could provide a useful liquid biopsy tool to improve cancer diagnostics and real-time surveillance of tumor evolution in patients to inform personalized therapy.

A robust microfluidic alternating-pull–push active digitization method based on pneumatic valve actuation enables reproducible and highly efficient digitization of small-volume samples for dPCR.