Liquid biopsy is extremely appealing in early diagnosis, prognosis, and precision treatment of cancer, as tissue biopsy is highly invasive, costly, and often infeasible to repeat. Extracellular vesicles (EVs), including exosomes, are emerging as a new paradigm of liquid biopsy for cancer diagnosis and monitoring response to therapy. Current affinity isolation and analysis methods rely on surface capture of EVs which is governed by the fundamental limits in mass transfer, surface binding reaction, and the boundary effects due to the hydrodynamic resistance at the liquid-solid interface. A major conceptual constraint in existing techniques is that they only partially addressed these fundamental factors. Here we report an innovative strategy for 3D nano-engineering of microfluidic chip to address the major limits in one device. This lithography-free engineering strategy exploits microfabricated channels to guide nanoparticle self-assembly, enabling simple, large-scale integration of self-assembled microelements with unique 3D nanostructures for biosensing. Using this method, we devised a nanoporous herringbone (nano-HB) chip that presents distinct advantages to address the aforementioned limits in one device, as it 1) effectively promotes microscale mass transfer; 2) increases the surface area and probe density to enhance binding efficiency and speed; and 3) reduces near-surface hydrodynamic resistance to promote particle-surface interactions for binding. Using the nano-HB chip, we demonstrated highly efficient (>80%) isolation of EVs purified from COLO-1, MCF-7, and SKOV3 cell lines spiked in healthy plasma. The nano-HB chip was further validated for elution and downstream analysis of captured EVs, i.e., targeted mRNA profiling by droplet digital PCR (ddPCR) and protein profiling by microplate ELISA. Assessed with spiked-in EV standards, the nano-HB chip was seen to afford an extremely low limit of detection of 10 μL-1 (200 vesicles per assay) and excellent compatibility with complex blood plasma. Such analytical capabilities enabled ultrasensitive and quantitative detection of low-level folate receptor alpha (FRα) positive exosomes in ovarian cancer patient plasma that are otherwise undetectable to standard assays. We demonstrated sensitive and specific detection of exosome subpopulations expressing CD24, EpCAM, and FRα proteins for non-invasive diagnosis of ovarian cancer using only 2 μL plasma. Comparative evaluation showed that our nano-HB chip greatly improves diagnostic power over the standard microplate ELISA and the combined nano-HB capture and ddPCR analysis of mRNAs. Our findings suggest exosomal FRα as a potential biomarker for early detection and monitoring of progression of ovarian cancer. Our technology would offer enabling exosome isolation and sensing abilities to facilitate liquid biopsy-based cancer diagnosis.

Citation Format: Peng Zhang, Xin Zhou, Yong Zeng. Ultrasensitive analysis of circulating exosomes for liquid biopsy-based cancer diagnosis using 3D nano-engineered microfluidic chips [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 415.