An Integrated Double-Filtration Microfluidic Device for Detection of Extracellular Vesicles from Urine for Bladder Cancer Diagnosis

Part of the Methods in Molecular Biology book series (MIMB, volume 1660)


Extracellular vesicles (EVs) are present in a variety of bodily fluids and they play an important role in cellular communications and signal transduction mechanisms. Studies have shown that the number of EVs and EV-associated biomarkers (i.e., proteins, nucleic acids and lipids) can be used to aid clinical diagnosis. Although ultracentrifugation is commonly used for EV isolation, it is not practical for clinical settings. Here, we developed an integrated double-filtration device that isolated and enriched EVs from urine, and subsequently detected/quantified EVs from urine via microchip ELISA. Results showed that the concentration of EVs was significantly elevated compared to healthy controls. Receiver operating characteristic analysis demonstrated that this integrated EV quantification device had a sensitivity of 81.3% at a specificity of 90% (16 bladder cancer patients and eight healthy controls). Thus, this integrated device shows great potential to supplement urine cytology for diagnosis of bladder cancer in point-of-care (POC) settings.

Key words

Filtration Extracellular vesicles (EVs) Microchip ELISA Bladder cancer Diagnostics 



Dr. Wang acknowledges the support from the Ministry of Science and Technology of the People’s Republic of China (2016YFC1101302) from China. Dr. Demirci would like to acknowledge R01 AI093282, R01 GM108584, R01 DE02497101, R01 AI081534, R21 Al113117, R21 Al110277, U54 EB015408, DOD LC150650 11976867, and Canary Center seed grant.

Competing financial interests: Dr. U. Demirci is a founder of, and has an equity interest in: (1) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, and (2) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions. U.D.’s interests were viewed and managed in accordance with the conflict of interest policies.


  1. 1.
    Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. CA Cancer J Clin 65(1):5–29CrossRefPubMedGoogle Scholar
  2. 2.
    National Cancer Institute (2016) Cancer of the urinary bladder. Accessed 1 May 2016
  3. 3.
    Kaufman DS, Shipley WU, Feldman AS (2009) Bladder cancer. Lancet 374(9685):239–249CrossRefPubMedGoogle Scholar
  4. 4.
    Tetu B, Tiguert R, Harel F, Fradet Y (2005) ImmunoCyt/uCyt+ improves the sensitivity of urine cytology in patients followed for urothelial carcinoma. Mod Pathol 18(1):83–89CrossRefPubMedGoogle Scholar
  5. 5.
    Jia S, Zocco D, Samuels ML, Chou MF, Chammas R, Skog J, Zarovni N, Momen-Heravi F, Kuo WP (2014) Emerging technologies in extracellular vesicle-based molecular diagnostics. Expert Rev Mol Diagn 14(3):307–321CrossRefPubMedGoogle Scholar
  6. 6.
    Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang HY, Thakur BK, Becker A, Hoshino A, Mark MT, Molina H, Xiang J, Zhang T, Theilen TM, Garcia-Santos G, Williams C, Ararso Y, Huang YJ, Rodrigues G, Shen TL, Labori KJ, Lothe IMB, Kure EH, Hernandez J, Doussot A, Ebbesen SH, Grandgenett PM, Hollingsworth MA, Jain M, Mallya K, Batra SK, Jarnagin WR, Schwartz RE, Matei I, Peinado H, Stanger BZ, Bromberg J, Lyden D (2015) Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17(6):816CrossRefPubMedGoogle Scholar
  7. 7.
    Franzen CA, Blackwell RH, Todorovic V, Greco KA, Foreman KE, Flanigan RC, Kuo PC, Gupta GN (2015) Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes. Oncogene 4:e163CrossRefGoogle Scholar
  8. 8.
    Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R (2015) Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523(7559):177–182CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nawaz M, Camussi G, Valadi H, Nazarenko I, Ekstrom K, Wang XQ, Principe S, Shah N, Ashraf NM, Fatima F, Neder L, Kislinger T (2014) The emerging role of extracellular vesicles as biomarkers for urogenital cancers. Nat Rev Urol 11(12):688–701CrossRefPubMedGoogle Scholar
  10. 10.
    Liga A, Vliegenthart ADB, Oosthuyzen W, Dear JW, Kersaudy-Kerhoas M (2015) Exosome isolation: a microfluidic road-map. Lab Chip 15(11):2388–2394CrossRefPubMedGoogle Scholar
  11. 11.
    Sunkara V, Woo HK, Cho YK (2016) Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics. Analyst 141(2):371–381CrossRefPubMedGoogle Scholar
  12. 12.
    Yoshioka Y, Kosaka N, Konishi Y, Ohta H, Okamoto H, Sonoda H, Nonaka R, Yamamoto H, Ishii H, Mori M, Furuta K, Nakajima T, Hayashi H, Sugisaki H, Higashimoto H, Kato T, Takeshita F, Ochiya T (2014) Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. Nat Commun 5:3591CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wang S, Xu F, Demirci U (2010) Advances in developing HIV-1 viral load assays for resource-limited settings. Biotechnol Adv 28(6):770–781CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wang SQ, Inci F, De Libero G, Singhal A, Demirci U (2013) Point-of-care assays for tuberculosis: role of nanotechnology/microfluidics. Biotechnol Adv 31(4):438–449CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wang S, Sarenac D, Chen MH, Huang SH, Giguel FF, Kuritzkes DR, Demirci U (2012) Simple filter microchip for rapid separation of plasma and viruses from whole blood. Int J Nanomedicine 7:5019–5028PubMedPubMedCentralGoogle Scholar
  16. 16.
    Wang S, Tasoglu S, Chen PZ, Chen M, Akbas R, Wach S, Ozdemir CI, Gurkan UA, Giguel FF, Kuritzkes DR, Demirci U (2014) Micro-a-fluidics ELISA for rapid CD4 cell count at the point-of-care. Sci Rep 4:3796CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wang S, Zhao X, Khimji I, Akbas R, Qiu W, Edwards D, Cramer DW, Ye B, Demirci U (2011) Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care. Lab Chip 11(20):3411–3418CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of MedicineZhejiang UniversityHangzhouChina
  2. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious DiseasesHangzhouChina
  3. 3.Institute for Translational MedicineZhejiang UniversityHangzhouChina
  4. 4.Department of Radiology, Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early DetectionStanford School of MedicinePalo AltoUSA
  5. 5.Department of Electrical EngineeringStanford UniversityStanfordUSA

Personalised recommendations