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Strand displacement-triggered G-quadruplex/rolling circle amplification strategy for the ultra-sensitive electrochemical sensing of exosomal microRNAs


Emerging evidence suggests that exosomal microRNAs are potential biomarkers for the early diagnosis and prognostic assessment of tumor. Here, we design a strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy for highly specific and sensitive electrochemical sensing of exosomal microRNAs. In the presence of exosomal miRNA-21, a locked nucleic acid (LNA)-labeled toehold mediated strand displacement reaction (TMSDR) is initiated, releasing output P2 to trigger the subsequent RCA reaction by hybridizing with the C-rich circular template. Then the obtained G-rich RCA products can bind to the probe anchored on the surface of gold electrode and generate G-quadruplex conformations. Based on the TMSDR-triggered G-quadruplex/RCA strategy, the detection limit of this electrochemical biosensor is down to 2.75 fM. Moreover, our biosensor exhibits excellent repeatability, stability, and high consistency compared to RT-PCR for clinical detection. In conclusion, this assay is expected to provide a hopeful strategy for the early non-invasive diagnosis and prognostic estimation of cancer.

Schematic illustration of electrochemical sensing of exosomal microRNAs based on strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy.

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  1. 1.

    Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 10:472–484

  2. 2.

    Sorbara L, Srivastava S (2019) Liquid biopsy: a holy grail for cancer detection. Biomark Med 13:991–994

  3. 3.

    Hannafon BN, Ding WQ (2013) Intercellular communication by exosome-derived microRNAs in cancer. Int J Mol Sci 14:14240–14269

  4. 4.

    He X, Ou C (2017) Exosome-derived microRNAs in cancer progression: angel or devil? J Thorac Dis 9:3440–3442

  5. 5.

    Tsui NB, Ng EK, Lo YM (2002) Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem 48:1647–1653

  6. 6.

    Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem 285:17442–17452

  7. 7.

    Yoshikawa M, Iinuma H, Umemoto Y, Yanagisawa T, Matsumoto A, Jinno H (2018) Exosome-encapsulated microRNA-223-3p as a minimally invasive biomarker for the early detection of invasive breast cancer. Oncol Lett 15:9584–9592

  8. 8.

    Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH (2009) Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer 10:42–46

  9. 9.

    Taylor DD, Gercel-Taylor C (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 110:13–21

  10. 10.

    Thomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, Rao TN, Winnay JN, Garcia-Martin R, Grinspoon SK, Gorden P, Kahn CR (2017) Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature 542:450–455

  11. 11.

    Ach RA, Wang H, Curry B (2008) Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods. BMC Biotechnol 8:69

  12. 12.

    Kim SW, Li Z, Moore PS, Monaghan AP, Chang Y, Nichols M, John B (2010) A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids Res 38:e98

  13. 13.

    Pritchard CC, Cheng HH, Tewari M (2012) MicroRNA profiling: approaches and considerations. Nat Rev Genet 13:358–369

  14. 14.

    Zhang X, Liu C, Pei Y, Song W, Zhang S (2019) Preparation of a novel Raman probe and its application in the detection of circulating tumor cells and Exosomes. ACS Appl Mater Interfaces 11:28671–28680

  15. 15.

    Wang Y, Li Z, Liu M, Xu J, Hu D, Lin Y, Li J (2017) Multiple-targeted graphene-based nanocarrier for intracellular imaging of mRNAs. Anal Chim Acta 983:1–8

  16. 16.

    Li XY, Cui YX, Du YC, Tang AN, Kong DM (2019) Label-free telomerase detection in single cell using a Five-Base telomerase product-triggered exponential rolling circle amplification strategy. Acs Sensors 4:1090–1096

  17. 17.

    Zhou L, Wang Y, Yang C, Xu H, Luo J, Zhang W, Tang X, Yang S, Fu W, Chang K, Chen M (2019) A label-free electrochemical biosensor for microRNAs detection based on DNA nanomaterial by coupling with Y-shaped DNA structure and non-linear hybridization chain reaction. Biosens Bioelectron 126:657–663

  18. 18.

    Zhao L, Sun R, He P, Zhang X (2019) Ultrasensitive detection of Exosomes by target-triggered three-dimensional DNA walking machine and exonuclease III-assisted electrochemical Ratiometric biosensing. Anal Chem 91:14773–14779

  19. 19.

    Li Y, Teng X, Zhang K, Deng R, Li J (2019) RNA Strand displacement responsive CRISPR/Cas9 system for mRNA sensing. Anal Chem 91:3989–3996

  20. 20.

    Jiang HX, Liang ZZ, Ma YH, Kong DM, Hong ZY (2016) G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection. Anal Chim Acta 943:114–122

  21. 21.

    Deng R, Zhang K, Li J (2017) Isothermal amplification for MicroRNA detection: from the test tube to the cell. Acc Chem Res 50:1059–1068

  22. 22.

    Yi L, Qinli P, Junlong L, Lili Z, Yiyi T, Yuxia L, Wen Y, Guoming X (2017) An "off-on" fluorescent switch assay for microRNA using nonenzymatic ligation-rolling circle amplification. Mikrochim Acta 184:4323

  23. 23.

    Du YC, Zhu YJ, Li XY, Kong DM (2018) Amplified detection of genome-containing biological targets using terminal deoxynucleotidyl transferase-assisted rolling circle amplification. Chem Commun 54:682–685

  24. 24.

    Sen D, Gilbert W (1988) Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature 334:364–366

  25. 25.

    Qian Y, Fan TT, Wang P, Zhang X, Luo JJ, Zhou FY, Yao Y, Liao XJ, Li YH, Gao FL (2017) A novel label-free homogeneous electrochemical immunosensor based on proximity hybridization-triggered isothermal exponential amplification induced G-quadruplex formation. Sensor Actuat B-Chem 248:187–194

  26. 26.

    Gao FL, Fan TT, Wu J, Liu S, Du Y, Yao Y, Zhou FY, Zhang Y, Liao XJ, Geng DQ (2017) Proximity hybridization triggered hemin/G-quadruplex formation for construction a label-free and signal-on electrochemical DNA sensor. Biosens Bioelectron 96:62–67

  27. 27.

    Zou R, Zhang F, Chen C, Cai C (2019) An ultrasensitive guanine wire-based resonance light scattering method using G-quadruplex self-assembly for determination of microRNA-122. Mikrochim Acta 186:599

  28. 28.

    Zhang FT, Nie J, Zhang DW, Chen JT, Zhou YL, Zhang XX (2014) Methylene blue as a G-quadruplex binding probe for label-free homogeneous electrochemical biosensing. Anal Chem 86:9489–9495

  29. 29.

    Srinivas N, Ouldridge TE, Sulc P, Schaeffer JM, Yurke B, Louis AA, Doye JP, Winfree E (2013) On the biophysics and kinetics of toehold-mediated DNA strand displacement. Nucleic Acids Res 41:10641–10658

  30. 30.

    Li S, Liu X, Pang S, Lu R, Liu Y, Fan M, Jia Z, Bai H (2018) Voltammetric determination of DNA based on regulation of DNA strand displacement using an allosteric DNA toehold. Mikrochim Acta 185:433

  31. 31.

    Chen J, Zhang J, Wang K, Lin X, Huang L, Chen G (2008) Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-aminobenzenesulfonic acid-modified glassy carbon electrode. Anal Chem 80:8028–8034

  32. 32.

    Wegman DW, Ghasemi F, Stasheuski AS, Khorshidi A, Yang BB, Liu SK, Yousef GM, Krylov SN (2016) Achieving single-nucleotide specificity in direct quantitative analysis of multiple MicroRNAs (DQAMmiR). Anal Chem 88:2472–2477

  33. 33.

    Wen SW, Sceneay J, Lima LG, Wong CS, Becker M, Krumeich S, Lobb RJ, Castillo V, Wong KN, Ellis S, Parker BS, Moller A (2016) The biodistribution and immune suppressive effects of breast Cancer-derived Exosomes. Cancer Res 76:6816–6827

  34. 34.

    Yuan YH, Chi BZ, Wen SH, Liang RP, Li ZM, Qiu JD (2018) Ratiometric electrochemical assay for sensitive detecting microRNA based on dual-amplification mechanism of duplex-specific nuclease and hybridization chain reaction. Biosens Bioelectron 102:211–216

  35. 35.

    Patra S, Roy E, Madhuri R, Sharma PK (2015) Imprinted ZnO nanostructure-based electrochemical sensing of calcitonin: a clinical marker for medullary thyroid carcinoma. Anal Chim Acta 853:271–284

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This work was supported by the National Natural Science Foundation of China (Grant No. 81430053, 81972027), Chongqing Health Commission (2018QNXM049, 2019ZDXM025) and Medical pre-research project of the Army Medical University (2018XYY04).

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Correspondence to Kai Chang or Ming Chen.

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Tang, X., Wang, Y., Zhou, L. et al. Strand displacement-triggered G-quadruplex/rolling circle amplification strategy for the ultra-sensitive electrochemical sensing of exosomal microRNAs. Microchim Acta 187, 172 (2020).

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  • Exosomal microRNAs
  • Locked nucleic acid
  • Strand displacement
  • Rolling circle amplification
  • G-quadruplex
  • Biosensor