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

Nature Biomedical Engineering volume 3, pages 438–451 (2019)Cite this article

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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.

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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.

References

  1. Schwarzenbach, H., Nishida, N., Calin, G. A. & Pantel, K. Clinical relevance of circulating cell-free microRNAs in cancer. Nat. Rev. Clin. Oncol. 11, 145–156 (2014).

    Article  CAS  Google Scholar 

  2. Haber, D. A. & Velculescu, V. E. Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA. Cancer Discov. 4, 650–661 (2014).

    Article  CAS  Google Scholar 

  3. He, M. & Zeng, Y. Microfluidic exosome analysis toward liquid biopsy for cancer. J. Lab. Autom. 21, 599–608 (2016).

    Article  CAS  Google Scholar 

  4. Im, H. et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat. Biotechnol. 32, 490–495 (2014).

    Article  CAS  Google Scholar 

  5. Yanez-Mo, M. et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 4, 27066 (2015).

    Article  Google Scholar 

  6. Atay, S. et al. Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion. Proc. Natl Acad. Sci. USA 111, 711–716 (2014).

    Article  CAS  Google Scholar 

  7. Melo, S. A. et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523, 177–182 (2015).

    Article  CAS  Google Scholar 

  8. Zhang, P., He, M. & Zeng, Y. Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating. Lab Chip 16, 3033–3042 (2016).

    Article  CAS  Google Scholar 

  9. Squires, T. M., Messinger, R. J. & Manalis, S. R. Making it stick: convection, reaction and diffusion in surface-based biosensors. Nat. Biotechnol. 26, 417–426 (2008).

    Article  CAS  Google Scholar 

  10. Stroock, A. D. et al. Chaotic mixer for microchannels. Science 295, 647–651 (2002).

    Article  CAS  Google Scholar 

  11. Shang, Y., Zeng, Y. & Zeng, Y. Integrated microfluidic lectin barcode platform for high-performance focused glycomic profiling. Sci. Rep. 6, 20297 (2016).

    Article  CAS  Google Scholar 

  12. Kelley, S. O. et al. Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering. Nat. Nanotechnol. 9, 969–980 (2014).

    Article  CAS  Google Scholar 

  13. Soleymani, L., Fang, Z., Sargent, E. H. & Kelley, S. O. Programming the detection limits of biosensors through controlled nanostructuring. Nat. Nanotechnol. 4, 844–848 (2009).

    Article  CAS  Google Scholar 

  14. Bin, X., Sargent, E. H. & Kelley, S. O. Nanostructuring of sensors determines the efficiency of biomolecular capture. Anal. Chem. 82, 5928–5931 (2010).

    Article  CAS  Google Scholar 

  15. Wang, S. et al. Three-dimensional nanostructured substrates toward efficient capture of circulating tumor cells. Angew. Chem. 48, 8970–8973 (2009).

    Article  CAS  Google Scholar 

  16. Chen, G. D., Fachin, F., Fernandez-Suarez, M., Wardle, B. L. & Toner, M. Nanoporous elements in microfluidics for multiscale manipulation of bioparticles. Small. 7, 1061–1067 (2011).

    Article  CAS  Google Scholar 

  17. Chen, G. D., Fachin, F., Colombini, E., Wardle, B. L. & Toner, M. Nanoporous micro-element arrays for particle interception in microfluidic cell separation. Lab Chip 12, 3159–3167 (2012).

    Article  CAS  Google Scholar 

  18. Fachin, F., Chen, G. D., Toner, M. & Wardle, B. L. Integration of bulk nanoporous elements in microfluidic devices with application to biomedical diagnostics. J. Microelectromech. Syst. 20, 1428–1438 (2011).

    Article  CAS  Google Scholar 

  19. Zeng, Y. & Harrison, D. J. Self-assembled colloidal arrays as three-dimensional nanofluidic sieves for separation of biomolecules on microchips. Anal. Chem. 79, 2289–2295 (2007).

    Article  CAS  Google Scholar 

  20. Stott, S. L. et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc. Natl Acad. Sci. USA 107, 18392–18397 (2010).

    Article  CAS  Google Scholar 

  21. Wang, S. et al. Highly efficient capture of circulating tumor cells by using nanostructured silicon substrates with integrated chaotic micromixers. Angew. Chem. 50, 3084–3088 (2011).

    Article  CAS  Google Scholar 

  22. Singh, G., Pillai, S., Arpanaei, A. & Kingshott, P. Multicomponent colloidal crystals that are tunable over large areas. Soft Matter 7, 3290–3294 (2011).

    Article  CAS  Google Scholar 

  23. Nazemifard, N. et al. A systematic evaluation of the role of crystalline order in nanoporous materials on DNA separation. Lab Chip 12, 146–152 (2012).

    Article  CAS  Google Scholar 

  24. Adams, A. A. et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. J. Am. Chem. Soc. 130, 8633–8641 (2008).

    Article  CAS  Google Scholar 

  25. Forbes, T. P. & Kralj, J. G. Engineering and analysis of surface interactions in a microfluidic herringbone micromixer. Lab Chip 12, 2634–2637 (2012).

    Article  CAS  Google Scholar 

  26. Sheng, W. et al. Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip. Lab Chip 14, 89–98 (2014).

    Article  CAS  Google Scholar 

  27. Philipse, A. P. & Pathmamanoharan, C. Liquid permeation (and sedimentation) of dense colloidal hard-sphere packings. J. Colloid. Interface Sci. 159, 96–107 (1993).

    Article  CAS  Google Scholar 

  28. Frishfelds, V., Hellstrom, J. G. I. & Lundstrom, T. S. Flow-induced deformations within random packed beds of spheres. Transport Porous Med. 104, 43–56 (2014).

    Article  CAS  Google Scholar 

  29. Shao, H. et al. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat. Commun. 6, 6999 (2015).

    Article  CAS  Google Scholar 

  30. Kanwar, S. S., Dunlay, C. J., Simeone, D. M. & Nagrath, S. Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 14, 1891–1900 (2014).

    Article  CAS  Google Scholar 

  31. He, M., Crow, J., Roth, M., Zeng, Y. & Godwin, A. K. Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology. Lab Chip 14, 3773–3780 (2014).

    Article  CAS  Google Scholar 

  32. Reategui, E. et al. Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles. Nat. Commun. 9, 175 (2018).

    Article  Google Scholar 

  33. Wan, Y. et al. Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes.Nat. Biomed. Eng. 1, 0058 (2017).

    Article  Google Scholar 

  34. Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 4, 27031 (2015).

    Article  Google Scholar 

  35. Domcke, S., Sinha, R., Levine, D. A., Sander, C. & Schultz, N. Evaluating cell lines as tumour models by comparison of genomic profiles.Nat. Commun. 4, 2126 (2013).

    Article  Google Scholar 

  36. Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476 (2008).

    Article  CAS  Google Scholar 

  37. Davies, R. T. et al. Microfluidic filtration system to isolate extracellular vesicles from blood. Lab Chip 12, 5202–5210 (2012).

    Article  CAS  Google Scholar 

  38. Li, S., Zhao, J., Lu, P. & Xie, Y. Maximum packing densities of basic 3D objects. Chinese Sci. Bull. 55, 114–119 (2010).

    Article  Google Scholar 

  39. Zhao, Z., Yang, Y., Zeng, Y. & He, M. A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis. Lab Chip 16, 489–496 (2016).

    Article  CAS  Google Scholar 

  40. Chevillet, J. R. et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc. Natl Acad. Sci. USA 111, 14888–14893 (2014).

    Article  CAS  Google Scholar 

  41. Toffoli, G. et al. Overexpression of folate binding protein in ovarian cancers. Int. J. Cancer 74, 193–198 (1997).

    Article  CAS  Google Scholar 

  42. Kurosaki, A. et al. Serum folate receptor alpha as a biomarker for ovarian cancer: implications for diagnosis, prognosis and predicting its local tumor expression. Int. J. Cancer 138, 1994–2002 (2016).

    Article  CAS  Google Scholar 

  43. Leung, F., Dimitromanolakis, A., Kobayashi, H., Diamandis, E. P. & Kulasingam, V. Folate-receptor 1 (FOLR1) protein is elevated in the serum of ovarian cancer patients. Clin. Biochem. 46, 1462–1468 (2013).

    Article  CAS  Google Scholar 

  44. Yang, J., Choi, M. K., Kim, D. H. & Hyeon, T. Designed assembly and integration of colloidal nanocrystals for device applications. Adv. Mater. 28, 1176–1207 (2016).

    Article  CAS  Google Scholar 

  45. Kim, J., Li, Z. & Park, I. Direct synthesis and integration of functional nanostructures in microfluidic devices. Lab Chip 11, 1946–1951 (2011).

    Article  CAS  Google Scholar 

  46. Jeon, S., Malyarchuk, V., White, J. O. & Rogers, J. A. Optically fabricated three dimensional nanofluidic mixers for microfluidic devices. Nano Lett. 5, 1351–1356 (2005).

    Article  CAS  Google Scholar 

  47. Park, S. G., Lee, S. K., Moon, J. H. & Yang, S. M. Holographic fabrication of three-dimensional nanostructures for microfluidic passive mixing. Lab Chip 9, 3144–3150 (2009).

    Article  CAS  Google Scholar 

  48. Hu, Y. et al. Laser printing hierarchical structures with the aid of controlled capillary-driven self-assembly. Proc. Natl Acad. Sci. USA 112, 6876–6881 (2015).

    Article  CAS  Google Scholar 

  49. Cederquist, K. B. & Kelley, S. O. Nanostructured biomolecular detectors: pushing performance at the nanoscale. Curr. Opin. Chem. Biol. 16, 415–421 (2012).

    Article  CAS  Google Scholar 

  50. Vogel, N., Retsch, M., Fustin, C. A., Del Campo, A. & Jonas, U. Advances in colloidal assembly: the design of structure and hierarchy in two and three dimensions. Chem. Rev. 115, 6265–6311 (2015).

    Article  CAS  Google Scholar 

  51. Bast, R. C. Jr, Hennessy, B. & Mills, G. B. The biology of ovarian cancer: new opportunities for translation. Nat. Rev. Cancer 9, 415–428 (2009).

    Article  CAS  Google Scholar 

  52. Bodurka, D. C. et al. Reclassification of serous ovarian carcinoma by a 2-tier system: a Gynecologic Oncology Group study. Cancer 118, 3087–3094 (2012).

    Article  Google Scholar 

  53. Cheung, A. et al. Targeting folate receptor alpha for cancer treatment. Oncotarget 7, 52553–52574 (2016).

    PubMed  PubMed Central  Google Scholar 

  54. Kuijk, A., van Blaaderen, A. & Imhof, A. Synthesis of monodisperse, rodlike silica colloids with tunable aspect ratio. J. Am. Chem. Soc. 133, 2346–2349 (2011).

    Article  CAS  Google Scholar 

  55. Kastelowitz, N. & Yin, H. Exosomes and microvesicles: identification and targeting by particle size and lipid chemical probes. ChemBioChem 15, 923–928 (2014).

    Article  CAS  Google Scholar 

  56. Müllner, T., Unger, K. K. & Tallarek, U. Characterization of microscopic disorder in reconstructed porous materials and assessment of mass transport-relevant structural descriptors. New J. Chem. 40, 3993–4015 (2016).

    Article  Google Scholar 

  57. Warnes, G. R. et al. gplots: Various R Programming Tools for Plotting Data v.3.0.1.1 (CRAN, 2019); https://cran.r-project.org/web/packages/gplots/index.html

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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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Authors and Affiliations

  1. Ralph N Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, KS, USA

    Peng Zhang, Xin Zhou, Mei He, Yuqin Shang & Yong Zeng

  2. Department of Chemical Engineering, University of Kansas, Lawrence, KS, USA

    Mei He

  3. Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA

    Ashley L. Tetlow & Andrew K. Godwin

  4. University of Kansas Cancer Center, Kansas City, KS, USA

    Andrew K. Godwin & Yong Zeng

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Contributions

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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