Microchimica Acta

, Volume 183, Issue 9, pp 2555–2562 | Cite as

DNA aptamer selection and aptamer-based fluorometric displacement assay for the hepatotoxin microcystin-RR

  • Shijia Wu
  • Qi Li
  • Nuo Duan
  • Haile Ma
  • Zhouping WangEmail author
Original Paper


Microcystin-RR (MC-RR) is a highly acute hepatotoxin produced by cyanobacteria. It is harmful to both humans and the environment. A novel aptamer was identified by the systemic evolution of ligands by exponential enrichment (SELEX) method as a recognition element for determination of MC-RR in aquatic products. The graphene oxide (GO) SELEX strategy was adopted to generate aptamers with high affinity and specificity. Of the 50 aptamer candidates tested, sequence RR-33 was found to display high affinity and selectivity, with a dissociation constant of 45.7 ± 6.8 nM. Aptamer RR-33 therefore was used as the recognition element in a fluorometric assay that proceeds as follows: (1) Biotinylated aptamer RR-33 is immobilized on the streptavidinylated wells of a microtiterplate, and carboxyfluorescein (FAM) labelled complementary DNA is then allowed to hybridize. (2) After removal of excess (unbound) cDNA, sample containing MC-RR is added and incubated at 37 °C for 2 h. (3) Displaced free cDNA is washed away and fluorescence intensity measured at excitation/emission wavelengths of 490/515 nm. The calibration plot is linear in the 0.20 to 2.5 ng·mL−1 concentration range, and the limit of detection is 80 pg·mL−1. The results indicate that the GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins. The detection scheme was applied to the determination of MC-RR in (spiked) water, mussel and fish and gave recoveries between 91 and 98 %. The method compares favorably to a known ELISA. Conceivably, this kind of assay is applicable to other toxins for which appropriate aptamers are available.

Graphical abstract

The aptamer RR-33 specific for microcystin (MC-RR) was identified by using the graphene oxide (GO) SELEX process. This aptamer was used for determination of MC-RR by a fluorometric displacement assay.


Cyanobacteria SELEX Biotinylated aptamer Microplate assay FTIR Transmission electron microscopy PAGE Taq polymerase Exonuclease 



This work was partially supported by the National Science and Technology Support Program of China (2015BAD17B02-5), China Postdoctoral Science Foundation (2016T90430, 2015 M580402), the NSFC (31401576, 31401575), BK20140155, Collaborative innovation center of food safety and quality control in Jiangsu Province, and S&T Support Program of Jiangsu Province BE2013732.

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2016_1904_MOESM1_ESM.docx (207 kb)
ESM 1 (DOCX 206 kb)


  1. 1.
    Carmichael WW (1992) Cyanobacterial secondary metabolites the cyanotoxins. Appl Bacteriol 72:445–459CrossRefGoogle Scholar
  2. 2.
    Carmichael WW (1994) The toxins of cyanobacteria. Sci Am 270:78–86CrossRefGoogle Scholar
  3. 3.
    Chen J, Xie P, Zhang D, Ke Z, Yang H (2006) In situ studies on the bioaccumulation of microcystins in the phytoplanktivoros silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms. Aquaculture 261:1026–1038CrossRefGoogle Scholar
  4. 4.
    Xie L, Xie P, Guo L, Li L, Miyabara Y, Park HD (2005) Organ distribution and bioaccumulation of microcystins in fresh fish at different trophic levels from the eutrophic Lake Chaohu, China. Environ Toxicol 20:293–300CrossRefGoogle Scholar
  5. 5.
    Mestrovic V, Pavela-Vrancic A (2003) Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor. Biochimie 85:647–650CrossRefGoogle Scholar
  6. 6.
    Dawson RM (1998) The toxicology of microcystins. Toxicon 36:953–962CrossRefGoogle Scholar
  7. 7.
    Schmidtkunz C, Stich HB, Welsch T (2009) Improving the selectivity and confidence in the HPLC analysis of microcystins in lake sediments. J Liq Chromatogr Relat Technol 32:801–821CrossRefGoogle Scholar
  8. 8.
    Sano T, Takagi H, Nagano K, Nishikawa M, Kaya K (2011) Accurate LC-MS analyses for microcystins using per-N-15-labeled microcystins. Anal Bioanal Chem 399:2511–2516CrossRefGoogle Scholar
  9. 9.
    Neffling MR, Spoof L, Meriluoto J (2009) Rapid LC-MS detection of cyanobacterial hepatotoxins microcystins and nodularins-Comparison of columns. Anal Chim Acta 653:234–241CrossRefGoogle Scholar
  10. 10.
    Samdal IA, Ballot A, Lovberg KE, Miels CO (2014) Mutihapten approach leading to a sensitive ELISA with broad cross-reactivity to microcystins and nodularin. Environ Sci Technol 48:8035–8043CrossRefGoogle Scholar
  11. 11.
    Yu FY, Liu BH, Chou HN, Chu FS (2002) Development of a sensitive ELISA for the determination of microcystins in algae. J Agric Food Chem 50:4176–4182CrossRefGoogle Scholar
  12. 12.
    Dawan S, Kanatharana P, Wongkittisuksa B, Limbut W, Numnuam A, Limsakul C, Thavarungkul P (2011) Label-free capacitive immunosensors for ultra-trace detection based on the increase of immobilized antibodies on silver nanoparticles. Anal Chim Acta 699:232–241CrossRefGoogle Scholar
  13. 13.
    Hu CL, Gan N Q, Chen YY, Bi LJ, Zhang XE, Song LR (2009) Detection of microcystins in environmental samples using surface Plasmon resonance biosensor. Talanta 80: 407–410Google Scholar
  14. 14.
    Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510CrossRefGoogle Scholar
  15. 15.
    Ellington AD, Szostak JW (1990) In vitro selection of RNA that bind specific ligands. Nature 346:818–822CrossRefGoogle Scholar
  16. 16.
    Cruz-Aguado JA, Penner G (2008) Determination of Ochratoxin A with a DNA aptamer. J Agric Food Chem 56:10456–10461CrossRefGoogle Scholar
  17. 17.
    Cox JC, Ellington AD (2001) Automated selection of anti-protein aptamers. J Bioorgan Med Chem 9:2525–2531CrossRefGoogle Scholar
  18. 18.
    Duan N, Wu SJ, Chen XJ, Huang YK, Wang ZP (2012) Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus. J Agric Food Chem 60:4034–4038CrossRefGoogle Scholar
  19. 19.
    Mok W, Li YF (2008) Recent progress in nucleic acid aptamer-based biosensors and bioassays. Sensors 8:7050–7084CrossRefGoogle Scholar
  20. 20.
    Tang LH, Liu Y, Ali MM, Kang DK, Zhao WA, Li JH (2012) Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme. Anal Chem 84:4711–4717CrossRefGoogle Scholar
  21. 21.
    Wu SJ, Duan N, Shi Z, Fang CC, Wang ZP (2014) Simultaneous aptasensor for multiplex pathogenic bacteria detection based on multicolor upconversion nanoparticles labels. Anal Chem 86:3100–3107CrossRefGoogle Scholar
  22. 22.
    Wu SJ, Duan N, Wang ZP, Wang HX (2011) Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin a using upconversion nanoparticles as labels. Analyst 136:2306–2314CrossRefGoogle Scholar
  23. 23.
    Wang GK, Lu YF, Yan CL, Lu Y (2015) DNA-functionalization gold nanoparticles based fluorescence sensor for sensitive detection of Hg2+ in aqueous solution. Sensor Actuat B-Chem 211:1–6CrossRefGoogle Scholar
  24. 24.
    Ng A, Chinnappan R, Eissa S, Liu HC, Tlili C, Zourob M (2012) Selection, characterization, and biosensing application of high affinity congener-specific microcystin-targeting aptamers. Environ Sci Technol 46:10697–10703CrossRefGoogle Scholar
  25. 25.
    Park JW, Tatavarty R, Kim DW, Jung HT, Gu MB (2012) Immobilization-free screening of aptamers assisted by graphene oxide. Chem Commun 48:2071–2073CrossRefGoogle Scholar
  26. 26.
    Park JW, Lee SJ, Cho EJ, Kim J, Song JY, Gu MB (2014) An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening. Biosens Bioelectron 51:324–329CrossRefGoogle Scholar
  27. 27.
    Ye ZQ, Tan MQ, Wang G, Yuan JL (2004) Preparation, characterization, and time-resolved fluorometric application of silica-coated terbium(ill) fluorescent nanoparticles. Anal Chem 76:513–518CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • Shijia Wu
    • 1
  • Qi Li
    • 1
  • Nuo Duan
    • 1
  • Haile Ma
    • 2
  • Zhouping Wang
    • 1
    Email author
  1. 1.State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.School of Food and Biological EngineeringJiangsu UniversityZhenjiangChina

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