Microchimica Acta

, 186:765 | Cite as

An aptamer-based fluorometric zearalenone assay using a lighting-up silver nanocluster probe and catalyzed by a hairpin assembly

  • Na Yin
  • Shuai Yuan
  • Man Zhang
  • Jingyi Wang
  • Ye Li
  • Yuan Peng
  • Jialei Bai
  • Baoan Ning
  • Jun Liang
  • Zhixian GaoEmail author
Original Paper


An enzyme-free fluorometric assay is described for the determination of zearalenone (ZEN). The method combines (a) catalyzed hairpin assembly (CHA), (b) ultrahigh fluorescent light-up G-rich DNA sequences in proximity to silver nanoclusters (Ag NCs), and (c) the use of aptamers (Apt). In the presence of ZEN, the inhibit sequence (Inh) is released from the aptamer-trigger sequence (Apt-T) via the binding of ZEN and the aptamer of Apt-T. The free Apt-T acts as a switch that opens the hairpins H1 and H2 to generate H1-H2 complex. The released Apt-T is available to trigger the next round of CHA between H1 and H2. Finally, the hybridization between H1 and the Ag NCs probe (P) causes the G-rich sequence to be in close proximity to the dark Ag NCs encapsulated by P. This leads to highly efficient lighting up of the Ag NCs and the production of amplified fluorescence with excitation/emission peaks at 575/628 nm. Under the optimized conditions, a linear correlation was observed with concentrations ranging from 1.3 pg mL−1 to 100 ng mL−1, and the limit of detection was 0.32 pg mL−1 (at S/N = 3). The method was successfully validated by analyzing maize and beer for levels of ZEN after a simple sample preparation procedure.

Graphical abstract

Schematic of the assay. The inhibit sequence (Inh) is released from aptamer-trigger sequence (Apt-T) via binding of ZEN and aptamer. The free Apt-T triggers catalyzed hairpin assembly (CHA).G-rich DNA is in proximity to silver nanoclusters (Ag NCs) and fluorescence intensity increases to detect ZEN.


Fungaltoxin Catalyzed hairpin assembly Guanine-rich DNA Fluorescence Template Nanoprobe Wide analytical range Beer analysis Maize analysis 



This work was supported by the National Key Research and Development Program of China (No.2017YFC1200903, No.2017YFC1200905), the National Natural Science Foundation of China (No.81773482), the National Key R&D Program of China (grant number 2018YFC1602500) and the Key Research and Development Program of Tianjin (No.18YFZCNC01260).

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Supplementary material

604_2019_3984_MOESM1_ESM.docx (2.9 mb)
ESM 1 (DOCX 2.93 mb)


  1. 1.
    Taghdisi SM, Danesh NM, Ramezani M, Emrani AS, Abnous K (2018) Novel colorimetric Aptasensor for Zearalenone detection based on nontarget-induced Aptamer Walker, gold nanoparticles, and exonuclease-assisted recycling amplification. ACS Appl Mater Interfaces 10:12504–12509CrossRefGoogle Scholar
  2. 2.
    Goud KY, Hayat A, Satyanarayana M, Kumar VS, Catanante G, Gobi KV, Marty JL (2017) Aptamer-based zearalenone assay based on the use of a fluorescein label and a functional graphene oxide as a quencher. Microchim Acta 184:4401–4408CrossRefGoogle Scholar
  3. 3.
    Jiang X, Li X, Yang Z, Eremin SA, Zhang X (2016) Evaluation and optimization of three different immunoassays for rapid detection Zearalenone in fodders. Food Anal Methods 10:256–262CrossRefGoogle Scholar
  4. 4.
    Liu N, Nie DX, Zhao ZY, Meng XJ, Wu AB (2015) Ultrasensitive immunoassays based on biotin-streptavidin amplified system for quantitative determination of family zearalenones. Food Control 57:202–209CrossRefGoogle Scholar
  5. 5.
    Zhang F, Liu B, Liu G, Sheng W, Zhang Y, Liu Q, Wang S (2018) Novel magnetic nanobeads-based fluoroimmunoassays for zearalenone detection in cereals using protein G as the recognition linker. Sensors Actuators B Chem 270:149–157CrossRefGoogle Scholar
  6. 6.
    Sun Y, Hu X, Zhang Y, Yang J, Wang F, Wang Y, Deng R, Zhang G (2014) Development of an immunochromatographic strip test for the rapid detection of zearalenone in corn. J Agric Food Chem 62:16–21Google Scholar
  7. 7.
    Liu G, Han Z, Nie D, Yang JH, Zhao ZH, Zhang JB, Li HP, Liao YC, Song SQ, De Saeger S, Wu AB (2012) Rapid and sensitive quantitation of zearalenone in food and feed by lateral flow immunoassay. Food Control 27:200–205CrossRefGoogle Scholar
  8. 8.
    Niazi S, Wang X, Pasha I, Khan IM, Zhao S, Shoaib M, Wu S, Wang Z (2018) A novel bioassay based on aptamer-functionalized magnetic nanoparticle for the detection of zearalenone using time resolved-fluorescence NaYF4: Ce/Tb nanoparticles as signal probe. Talanta 186:97–103CrossRefGoogle Scholar
  9. 9.
    Chun HS, Choia EH, Chang H, Choi S, Eremin SA (2009) A fluorescence polarization immunoassay for the detection of zearalenone in corn. Anal Chim Acta 639:83–89CrossRefGoogle Scholar
  10. 10.
    He Q, Peng H, Yang J, Xu Z, Fan C, Sun Y (2017) QuEChERS extraction followed by enzyme-linked immunosorbent assay for determination of deoxynivalenol and zearalenone in cereals. Food Agric Immunol 28:1–19CrossRefGoogle Scholar
  11. 11.
    Wang YK, Zou Q, Sun JH, Wang HA, Sun X, Chen ZF, Yan YX (2015) Screening of single-stranded DNA (ssDNA) aptamers against a zearalenone monoclonal antibody and development of a ssDNA-based enzyme-linked oligonucleotide assay for determination of zearalenone in corn. J Agric Food Chem 63:36–41Google Scholar
  12. 12.
    Pei S, Lee W, Zhang G, Hu X, Eremin S, Zhang L (2013) Development of anti-zearalenone monoclonal antibody and detection of zearalenone in corn products from China by ELISA. Food Control 31:65–70CrossRefGoogle Scholar
  13. 13.
    Xu W, Qing Y, Chen S, Chen J, Qin Z, Qiu J, Li C (2017) Electrochemical indirect competitive immunoassay for ultrasensitive detection of zearalenone based on a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes and chitosan. Microchim Acta 184:3339–3347CrossRefGoogle Scholar
  14. 14.
    Dong GL, Pan YH, Wang YL, Ahmed S, Liu ZL, Peng DP, Yuan ZH (2018) Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linked immunosorbent assay. Food Chem 247:8–15CrossRefGoogle Scholar
  15. 15.
    Chen J, Zhang X, Cai S, Wu D, Chen M, Wang S, Zhang J (2014) A fluorescent Aptasensor based on DNA-Scaffolded silver-Nanocluster for Ochratoxin a detection. Biosens Bioelectron 57:226–231CrossRefGoogle Scholar
  16. 16.
    Li S, Wang J, Sheng W, Wen W, Gu Y, Wang S (2018) Fluorometric lateral flow immunochromatographic zearalenone assay by exploiting a quencher system composed of carbon dots and silver nanoparticles. Mikrochim Acta 185:388–397CrossRefGoogle Scholar
  17. 17.
    Chen J, Ji X, Tinnefeld P, He Z (2016) Multifunctional dumbbell-shaped DNA-Templated selective formation of fluorescent silver Nanoclusters or copper nanoparticles for sensitive detection of biomolecules. ACS Appl Mater Interfaces 8:86–94Google Scholar
  18. 18.
    Sharma J, Yeh HC, Yoo H, Werner JH, Martinez JS (2011) Silver nanocluster aptamers: in situ generation of intrinsically fluorescent recognition ligands for protein detection. Chem Commun 47:2294–2296CrossRefGoogle Scholar
  19. 19.
    Zhang J, Liu Y, Zhi X, Zhang C, Liu T, Cui D (2018) DNA-Templated silver Nanoclusters locate MicroRNAs in the nuclei of gastric Cancer cells. Nanoscale 10:11079–11090CrossRefGoogle Scholar
  20. 20.
    Yu JH, Choi S, Dickson RM (2009) Shuttle-based Fluorogenic silver-cluster biolabels. Angew Chem Int Ed 48:318–320CrossRefGoogle Scholar
  21. 21.
    New SY, Lee ST, Su XD (2016) DNA-Templated silver Nanoclusters: structural correlation and fluorescence modulation. Nanoscale 8:17729–17746CrossRefGoogle Scholar
  22. 22.
    Li JL, Ma JH, Zhang YC, Zhang ZL, He GW (2019) A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe. Microchim Acta 186:48CrossRefGoogle Scholar
  23. 23.
    Yeh HC, Sharma J, Han JJ, Martinez JS, Werner JH (2010) A DNA-silver Nanocluster probe that fluoresces upon hybridization. Nano Lett 10:3106–3110CrossRefGoogle Scholar
  24. 24.
    Pan M, Liang M, Sun J, Liu X, Wang F (2018) Lighting up fluorescent silver clusters via target-catalyzed hairpin assembly for amplified biosensing. Langmuir 34:14851–14857CrossRefGoogle Scholar
  25. 25.
    Peng Y, Li DX, Yuan R, Xiang Y (2018) A catalytic and dual recycling amplification ATP sensor based on target driven allosteric structure switching of aptamer beacons. Biosens Bioelectron 105:1–5CrossRefGoogle Scholar
  26. 26.
    Wang Y, Gan N, Zhou Y, Li TH, Hu FT, Cao YT, Chen YJ (2017) Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification. Biosens Bioelectron 97:100–106CrossRefGoogle Scholar
  27. 27.
    Chen X, Huang Y, Duan N, Wu S, Ma X, Xia Y, Zhu C, Jiang Y, Wang Z (2013) Selection and identification of ssDNA aptamers recognizing zearalenone. Anal Bioanal Chem 405:6573–6581CrossRefGoogle Scholar
  28. 28.
    Zhang P, Liu H, Li XC, Ma SZ, Men S, Wei H, Cui JJ, Wang HN (2017) A label-free fluorescent direct detection of live Salmonella typhimurium using cascade triple trigger sequences-regenerated strand displacement amplification and hairpin template-generated-scaffolded silver nanoclusters. Biosens Bioelectron 87:1044–1049CrossRefGoogle Scholar
  29. 29.
    Yeh HC, Sharma J, Shih Ie M, Vu DM, Martinez JS, Werner JH (2012) A fluorescence light-up Ag nanocluster probe that discriminates single-nucleotide variants by emission color. J Am Chem Soc 134:11550–11558CrossRefGoogle Scholar
  30. 30.
    Ge L, Sun X, Hong Q, Li F (2017) Ratiometric NanoCluster Beacon: a label-free and sensitive fluorescent DNA detection platform. ACS Appl Mater Interfaces 9:13102–13110CrossRefGoogle Scholar
  31. 31.
    Zhou ZX, Du Y, Dong SJ (2011) DNA-Ag nanoclusters as fluorescence probe for turn-on aptamer sensor of small molecules. Biosens Bioelectron 28:33–37CrossRefGoogle Scholar
  32. 32.
    Liu J (2014) DNA-stabilized, fluorescent, metal Nanoclusters for biosensor development. TrAC-Trend Anal Chem 58:99–111CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.State Key Laboratory of Food Nutrition and SafetyTianjin University of Science and TechnologyTianjinChina
  2. 2.Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food SafetyTianjin Institute of Environmental and Operational MedicineTianjinChina

Personalised recommendations