Microchimica Acta

, 186:796 | Cite as

A hybridization chain reaction based assay for fluorometric determination of exosomes using magnetic nanoparticles and both aptamers and antibody as recognition elements

  • Liangliang Shi
  • Li Ba
  • Ying Xiong
  • Gang PengEmail author
Original Paper


Exosomes represent a new generation of biomarkers for the early diagnosis of hepatic carcinoma. A fluorometric assay is presented that is based on the hybridization chain reaction and magnetic nanoparticles for the highly sensitive determination of exosome (from HepG2 cells). Antibody as the recognition element was modified on the surface of magnetic nanoparticles. Antibody was used to capture the exosome. The Probe 1 was consisted of aptamer sequence and trigger sequence. Aptamer sequence will bind with the surface protein of exosome. The trigger sequence will hybridize with the probe2 (FAM-labeled) and the probe3 (FAM-labeled). So the product of the hybridization chain reaction will present a strong fluorescence signal. The fluorescence product of hybridization chain reaction will link with magnetic nanoparticles through the ‘magnetic nanoparticles-antibody-exosome-aptamer’ structure. The product can be separated from the matrix due to the present of the magnetic nanoparticles. The excitation was set at 490 nm. The fluorescence value of the emission spectra at 519 nm was set as the signal response. The linear range of this assay is from 1000 to 107 particles·mL−1. The detection limit is 100 particles·mL−1. This assay was applied to the determination of exosome from the hepatic carcinoma cells.

Graphical abstract

In the presence of exosmes, the hybridization chain reaction was triggered and strong green fluorescence will be produced. Moreover, the magnetic particles can separate the products from the matrix.


Biomarkers Tumor Hepatic carcinoma HepG2 cells Protein Sensor Diagnosis 



This work was supported by Hubei Natural Science Fund (2015CFB668) and Independent Innovation Fund of Huazhong University of Science and Technology (0118530318).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

604_2019_3823_MOESM1_ESM.docx (387 kb)
ESM 1 (DOCX 387 kb)


  1. 1.
    Oliveira SD, Houseright RA, Graves AL, Golenberg N, Korte BG, Miskolci V, Huttenlocher A (2019) Metformin modulates innate immune-mediated inflammation and early progression of NAFLD-associated hepatocellular carcinoma in zebrafish. J Hepatol 70:710–721CrossRefGoogle Scholar
  2. 2.
    Altekruse SF, McGlynn KA, Reichman ME (2009) Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol 27:1485–1491CrossRefGoogle Scholar
  3. 3.
    Zeng J, Yin P, Tan Y, Dong L, Hu C, Huang Q, Lu X, Wang H, Xu G (2014) Metabolomics study of hepatocellular carcinoma: discovery and validation of serum potential biomarkers by using capillary electrophoresis-mass spectrometry. J Proteome Res 13:3420–3431CrossRefGoogle Scholar
  4. 4.
    Rolfo C, Castiglia M, Hong D, Alessandro R, Mertens I, Baggerman G, Zwaenepoel K, Gil-Bazo I, Passiglia F, Carreca AP, Taverna S, Vento R, Santini D, Peeters M, Russo A, Pauwels P (2014) Liquid biopsies in lung cancer: the new ambrosia of researchers. Biochim Biophys Acta 1846:539–546PubMedGoogle Scholar
  5. 5.
    Lianidou E, Pantel K (2019) Liquid biopsies. Genes Chromosom Cancer 58:219–232CrossRefGoogle Scholar
  6. 6.
    Buckanovich RJ, Sasaroli D, O'Brien-Jenkins A, Botbyl J, Hammond R, Katsaros D, Sandaltzopoulos R, Liotta LA, Gimotty PA, Coukos G (2007) Tumor vascular proteins as biomarkers in ovarian cancer. J Clin Oncol 25:852–861CrossRefGoogle Scholar
  7. 7.
    Leary RJ, Kinde I, Diehl F, Schmidt K, Clouser C, Duncan C, Antipova A, Lee C, McKernan K, Vega FM, Kinzler KW, Vogelstein B, Diaz JLA, Velculescu VE (2010) Development of personalized tumor biomarkers using massively parallel sequencing. Sci Transl Med (20):1–7CrossRefGoogle Scholar
  8. 8.
    Lianidou ES, Markou A (2011) Circulating tumor cells as emerging tumor biomarkers in breast cancer. Clin Chem Lab Med 49:1579–1590CrossRefGoogle Scholar
  9. 9.
    Kim JU, Shariff MI, Crossey MM, Gomez-Romero M, Holmes E, Cox IJ, Fye HK, Njie R, Taylor-Robinson SD (2016) Hepatocellular carcinoma: review of disease and tumor biomarkers. World J Hepatol 8:471–484CrossRefGoogle Scholar
  10. 10.
    Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579CrossRefGoogle Scholar
  11. 11.
    Sasaki R, Kanda T, Yokosuka O, Kato N, Matsuoka S, Moriyama M (2019) Exosomes and hepatocellular carcinoma: from bench to bedside. Int J Mol Sci 20:1406CrossRefGoogle Scholar
  12. 12.
    Fu M, Gu J, Jiang P, Qian H, Xu W, Zhang X (2019) Exosomes in gastric cancer: roles, mechanisms, and applications. Mol Cancer 18:41CrossRefGoogle Scholar
  13. 13.
    Yao Z, Jia X, Megger DA, Chen J, Liu Y, Li J, Sitek B, Yuan Z (2019) Label-free proteomic analysis of exosomes secreted from THP-1-derived macrophages treated with IFN-alpha identifies antiviral proteins enriched in exosomes. J Proteome Res 18:855–864CrossRefGoogle Scholar
  14. 14.
    Li Q, Tofaris GK, Davis JJ (2017) Concentration-normalized electroanalytical assaying of Exosomal markers. Anal Chem 89:3184–3190CrossRefGoogle Scholar
  15. 15.
    Gorodkiewicz E, Lukaszewski Z (2018) Recent Progress in surface Plasmon resonance biosensors (2016 to Mid-2018). Biosensors-Basel 8:132CrossRefGoogle Scholar
  16. 16.
    Xia YK, Liu MM, Wang LL, Yan A, He WH, Chen M, Lan JM, Xu JX, Guan LH, Chen JH (2017) A visible and colorimetric aptasensor based on DNA-capped single-walled carbon nanotubes for detection of exosomes. Biosens Bioelectron 92:8–15CrossRefGoogle Scholar
  17. 17.
    Shin H, Jeong H, Park J, Hong S, Choi Y (2018) Correlation between cancerous exosomes and protein markers based on surface-enhanced Raman spectroscopy (SERS) and principal component analysis (PCA). ACS SENSORS 3:2637–2643CrossRefGoogle Scholar
  18. 18.
    Carney RP, Hazari S, Colquhoun M, Di T, Hwang B, Mulligan MS, Bryers JD, Girda E, Leiserowitz GS, Smith ZJ, Lam KS (2017) Multispectral optical tweezers for biochemical fingerprinting of CD9-positive exosome subpopulations. Anal Chem 89:5357–5363CrossRefGoogle Scholar
  19. 19.
    Busatto S, Giacomini A, Montis C, Ronca R, Bergese P (2018) Uptake profiles of human serum exosomes by murine and human tumor cells through combined use of colloidal Nanoplasmonics and flow Cytofluorimetric analysis. Anal Chem 90:7855–7861CrossRefGoogle Scholar
  20. 20.
    Rupert DLM, Mapar M, Shelke GV, Norling K, Elmeskog M, Lotvall JO, Block S, Bally M, Agnarsson B, Hook F (2018) Effective refractive index and lipid content of extracellular vesicles revealed using optical waveguide scattering and fluorescence microscopy. Langmuir 34:8522–8531CrossRefGoogle Scholar
  21. 21.
    Zhang K, Gan N, Hu F, Chen X, Li T, Cao J (2018) Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction. Microchim Acta 185:181CrossRefGoogle Scholar
  22. 22.
    Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9:537–550CrossRefGoogle Scholar
  23. 23.
    Hong M, Wang M, Wang J, Xu X, Lin Z (2017) Ultrasensitive and selective electrochemical biosensor for detection of mercury (II) ions by nicking endonuclease-assisted target recycling and hybridization chain reaction signal amplification. Biosens Bioelectron 94:19–23CrossRefGoogle Scholar
  24. 24.
    Yin J, Gan P, Zhou F, Wang J (2014) Sensitive detection of transcription factors using near-infrared fluorescent solid-phase rolling circle amplification. Anal Chem 86:2572–2579CrossRefGoogle Scholar
  25. 25.
    Shen R, Zou L, Wu S, Li T, Wang J, Liu J, Ling L (2019) A novel label-free fluorescent detection of histidine based upon Cu(2+)-specific DNAzyme and hybridization chain reaction. Spectrochim Acta A Mol Biomol Spectrosc 213:42–47CrossRefGoogle Scholar
  26. 26.
    Wu C, Cansiz S, Zhang L, Teng IT, Qiu L, Li J, Liu Y, Zhou C, Hu R, Zhang T, Cui C, Cui L, Tan W (2015) A nonenzymatic hairpin DNA Cascade reaction provides high signal gain of mRNA imaging inside live cells. J Am Chem Soc 137:4900–4903CrossRefGoogle Scholar
  27. 27.
    Lu AH, Salabas EL, Schuth F (2007) Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem 46:1222–1244CrossRefGoogle Scholar
  28. 28.
    Hao R, Xing R, Xu Z, Hou Y, Gao S, Sun S (2010) Synthesis, functionalization, and biomedical applications of multifunctional magnetic nanoparticles. Adv Mater 22:2729–2742CrossRefGoogle Scholar
  29. 29.
    Yang Y, Li C, Shi H, Chen T, Wang Z, Li G (2019) A pH-responsive bioassay for paper-based diagnosis of exosomes via mussel-inspired surface chemistry. Talanta 192:325–330CrossRefGoogle Scholar
  30. 30.
    Tian YF, Ning CF, He F, Yin BC, Ye BC (2018) Highly sensitive detection of exosomes by SERS using gold nanostar@ Raman reporter@ nanoshell structures modified with a bivalent cholesterollabeled DNA anchor. Analyst 143(20):4915–4922CrossRefGoogle Scholar
  31. 31.
    Zhu L, Wang K, Cui J, Liu H, Bu X, Ma H, Wang W, Gong H, Lausted C, Hood L et al (2014) Label-free quantitative detection of tumor-derived exosomes through surface Plasmon resonance imaging. Anal Chem 86(17):8857–8864CrossRefGoogle Scholar
  32. 32.
    Xu H, Liao C, Zuo P, Liu Z, Ye B-C (2018) Magnetic-based microfluidic device for on-Chip isolation and detection of tumor-derived exosomes. Anal Chem 90(22):13451–13458CrossRefGoogle Scholar
  33. 33.
    Zhang Q, Wang F, Zhang H, Zhang Y, Liu M, Liu Y (2018) Universal Ti3C2 MXenes based self-standard Ratiometric fluorescence resonance energy transfer platform for highly sensitive detection of exosomes. Anal Chem 90(21):12737–12744CrossRefGoogle Scholar
  34. 34.
    Boriachek K, Masud MK, Palma C, Phan HP, Yamauchi Y, Hossain MSA, Nguyen NT, Salomon C, Shiddiky MJA (2019) Avoiding pre-isolation step in exosome analysis: direct isolation and sensitive detection of exosomes using gold-loaded Nanoporous ferric oxide Nanozymes. Anal Chem 91(6):3827–3834CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Cancer Center, Union Hospital, Tongji Medical CollegeHuazhong University of science and TechnologyHubeiChina

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