Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip

Abstract

The performance of current microfluidic methods for exosome detection is constrained by boundary conditions, as well as fundamental limits to microscale mass transfer and interfacial exosome binding. Here, we show that a microfluidic chip designed with self-assembled three-dimensional herringbone nanopatterns can detect low levels of tumour-associated exosomes in plasma (10 exosomes μl−1, or approximately 200 vesicles per 20 μl of spiked sample) that would otherwise be undetectable by standard microfluidic systems for biosensing. The nanopatterns promote microscale mass transfer, increase surface area and probe density to enhance the efficiency and speed of exosome binding, and permit drainage of the boundary fluid to reduce near-surface hydrodynamic resistance, thus promoting particle–surface interactions for exosome binding. We used the device for the detection—in 2 μl plasma samples from 20 ovarian cancer patients and 10 age-matched controls—of exosome subpopulations expressing CD24, epithelial cell adhesion molecule and folate receptor alpha proteins, and suggest exosomal folate receptor alpha as a potential biomarker for early detection and progression monitoring of ovarian cancer. The nanolithography-free nanopatterned device should facilitate the use of liquid biopsies for cancer diagnosis.

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Fig. 1: MINDS.
Fig. 2: Fluidic characterization of nano-HB chips.
Fig. 3: 3D-engineered nano-HB chip affords efficient immunocapture of exosomes.
Fig. 4: Ultrasensitive detection of exosomes with the nano-HB chip.
Fig. 5: Clinical profiling of circulating exosomes for the diagnosis of ovarian cancer.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request.

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Acknowledgements

We thank the microfabrication core facility at the KU COBRE Center for Molecular Analysis of Disease Pathways for device fabrication, and the KU Cancer Center’s Biospecimen Repository Core Facility, funded in part by the National Cancer Institute Cancer Center Support Grant (P30 CA168524) for providing clinical plasma samples, T. Meyer (KUMC) for technical support with immunohistochemistry, R. Madan (KUMC) for histopathology review, and H. Pathak (KUCC) for technical guidance with cell line-derived exosome isolations. This study was supported by 1R21CA186846, 1R21CA207816, 1R21EB024101, 1R33CA214333 and P20GM103638 from the NIH. P.Z. was supported by the postdoc award from the Kansas IDeA Network of Biomedical Research Excellence under the grant P20GM103418 from NIH/NIGMS. A.K.G. is the Chancellors Distinguished Chair in Biomedical Sciences Endowed Professor.

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Y.Z. conceived and supervised the project. P.Z. and Y.Z. designed the research. P.Z. performed the technology development, microfluidic analysis and microscopic imaging. X.Z. and P.Z. conducted mRNA profiling. M.H. contributed to numerical simulation. Y.S. isolated exosomes from clinical samples, and conducted western blot and some NTA analyses. A.L.T. isolated exosomes from cell culture media and helped with the immunohistochemistry. A.K.G. provided clinical samples and assisted in clinical studies. P.Z., X.Z., M.H., Y.S., A.K.G. and Y.Z. analysed the data. P.Z. and Y.Z. wrote the manuscript. All authors edited the manuscript.

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Correspondence to Yong Zeng.

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Zhang, P., Zhou, X., He, M. et al. Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip. Nat Biomed Eng 3, 438–451 (2019). https://doi.org/10.1038/s41551-019-0356-9

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