Analytical and Bioanalytical Chemistry

, Volume 408, Issue 30, pp 8593–8601 | Cite as

Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification

  • Juan TangEmail author
  • Yapei Huang
  • Huiqiong Liu
  • Cengceng Zhang
  • Dianping TangEmail author
Research Paper
Part of the following topical collections:
  1. Isothermal Nucleic Acid Amplification in Bioanalysis


A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B1 (AFB1) in foodstuff. The system consisted of anti-AFB1 antibody labeled DNA1 (Ab-DNA1), AFB1–bovine serum albumin (BSA)-conjugated DNA2 (AFB1-DNA2), and methylene blue functionalized hairpin DNA. Owing to a specific antigen–antibody reaction between anti-AFB1 and AFB1–BSA, the immunocomplex formed assisted the proximity hybridization of DNA1 with DNA2, thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB1, the analyte competed with AFB1-DNA2 for the conjugated anti-AFB1 on the Ab-DNA1, subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB1 in a dynamic working range of 0.01–30 ng mL-1 with a detection limit of 4.8 pg mL-1. In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB1-spiked peanut samples, giving results that matched well with those obtained with a commercial AFB1 ELISA kit.


Electrochemical immunoassay Aflatoxin B1 Proximity ligation reaction Omega-like DNA junction Isothermal nucleic acid cycling amplification 



Support by the National Natural Science Foundation of China (grant nos. 21505060, 41176079, and 21475025), the National Science Foundation of Fujian Province (grant no. 2014J07001), and the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT15R11) is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2016_9343_MOESM1_ESM.pdf (107 kb)
ESM 1 (PDF 106 kb)


  1. 1.
    Raiola A, Tenore G, Manyes L, Meca G. Ritieni. Risk analysis of main mycotoxins occurring in food for children: an overview. Food Chem Toxicol. 2015;84:169–80.CrossRefGoogle Scholar
  2. 2.
    Milani J, Maleki G. Effects of processing on mycotoxin stability in cereals. J Sci Food Agric. 2014;94:2372–5.CrossRefGoogle Scholar
  3. 3.
    Flores-Flores M, Lizarraga E, Lopez de Cerain A, Gonzalez-Penas E. Presence of mycotoxins in animal milk: a review. Food Control. 2015;53:163–76.CrossRefGoogle Scholar
  4. 4.
    Lin Y, Zhou Q, Lin Y, Tang D, Chen G, Tang D. Simple and sensitive detection of aflatoxin B1 within five minute using a non-conventional competitive immunosensing mode. Biosens Bioelectron. 2015;74:680–6.CrossRefGoogle Scholar
  5. 5.
    Vidal J, Bonel L, Ezquerra A, Hernandez S, Bertolin J, Cubel C, et al. Electrochemical affinity biosensors for detection of mycotoxins: a review. Biosens Bioelectron. 2013;49:146–58.CrossRefGoogle Scholar
  6. 6.
    Wang H, Zhang Y, Chu Y, Ma H, Li Y, Wu D, et al. Disposable competitive-type immunoassay for determination of aflatoxin B1 via detection of copper ions released from Cu-apatite. Talanta. 2016;147:556–60.CrossRefGoogle Scholar
  7. 7.
    Goryacheva I. Contemporary trends in the development of immunochemical methods for medical analysis. J Anal Chem. 2015;70:903–14.CrossRefGoogle Scholar
  8. 8.
    Zhao F, Hu C, Wang H, Zhao L, Yang Z. Development of a MAb-based immunoassay for the simultaneous determination of O, O-diethyl and O, O-dimethyl organophosphorus pesticides in vegetable and fruit samples pretreated with QuEChERS. Anal Bioanal Chem. 2015;407:8959–70.CrossRefGoogle Scholar
  9. 9.
    Lin Y, Zhou Q, Lin Y, Tang D, Niessner R, Knopp D. Enzymatic hydrolysate-induced displacement reaction with multifunctional silica beads doped with horseradish peroxidase–thionine conjugate for ultrasensitive electrochemical immunoassay. Anal Chem. 2015;87:8531–40.CrossRefGoogle Scholar
  10. 10.
    Liu J, Lu C, Liu B, Yu F. Development of novel monoclonal antibodies-based ultransensitive enzyme-linked assay and rapid immunochromatographic strip. Food Control. 2016;59:700–7.CrossRefGoogle Scholar
  11. 11.
    Chauhan R, Singh J, Solanki P, Manaka T, Iwamoto M, Basu T, et al. Label-free piezoelectric immunosensor decorated with gold nanoparticles: kinetic analysis and biosensing application. Sens Actuators B. 2016;222:804–14.CrossRefGoogle Scholar
  12. 12.
    Anfossi L, Di Nardo F, Giovannoli C, Passini C, Baggiani C. Enzyme immunoassay for monitoring aflatoxins in eggs. Food Control. 2015;57:115–21.CrossRefGoogle Scholar
  13. 13.
    Zhu R, Zhao Z, Wang J, Bai B, Wu A, Yan L, et al. A simple sample pretreatment method for multi-mycotoxin determination in eggs by liquid chromatography tandem mass spectrometry. J Chromatogr A. 2015;1417:1–7.CrossRefGoogle Scholar
  14. 14.
    Wang X, Pauli J, Niessner R, Resch-Genger U, Knopp D. Gold nanoparticle-catalyzed uranine reduction for signal amplification in fluorescent assays for melamine and aflatoxin B1. Analyst. 2015;140:7305–12.CrossRefGoogle Scholar
  15. 15.
    Zhao Y, Yang Y, Luo Y, Yang X, Li M, Song Q. Double detection of mycotoxins based on SERS labels embedded Ag@Au core-shell nanoparticles. ACS Appl Mater Interfaces. 2015;7:21780–6.CrossRefGoogle Scholar
  16. 16.
    Adornetto G, Fabiani L, Volpe G, Stefano A, Martini S, Nenna R, et al. An electrochemical immunoassay for the screening of celiac disease in saliva samples. Anal Bioanal Chem. 2015;407:7189–96.CrossRefGoogle Scholar
  17. 17.
    Tang D, Su B, Tang J, Ren J, Chen G. Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads. Anal Chem. 2010;82:1527–34.CrossRefGoogle Scholar
  18. 18.
    Tang J, Tang D, Niessner R, Knopp D. A novel strategy for ultra-sensitive electrochemical immunoassay of biomarkers by coupling multifunctional iridium oxide (IrOx) nanospheres with catalytic recycling of self-produced reactants. Anal Bioanal Chem. 2011;400:2041–51.CrossRefGoogle Scholar
  19. 19.
    Zhang Y, Huang Y, Jiang J, Shen G, Yu R. Electrochemical aptasensor based on proximity-dependent surface hybridization assay for single-step, reusable, sensitive protein detection. J Am Chem Soc. 2007;129:15448–9.CrossRefGoogle Scholar
  20. 20.
    Nie H, Liu S, Yu R, Jiang J. Phospholipid-coated carbon nanotubes as sensitive electrochemical labels with controlled-assembly-mediated signal transduction for magnetic separation immunoassay. Angew Chem Int Ed. 2009;48:9862–6.CrossRefGoogle Scholar
  21. 21.
    Akhavan-Tafti H, Binger D, Blackwood J, Chen Y, Creager R, de Silva R, et al. A homogeneous chemiluminescent immunoassay method. J Am Chem Soc. 2013;135:4191–4.CrossRefGoogle Scholar
  22. 22.
    Tang D, Lin Y, Zhou Q, Lin Y, Li P, Niessner R, et al. Low-cost and highly sensitive immunosensing platform for aflatoxins using one-step competitive displacement reaction mode and portable glucometer-based detection. Anal Chem. 2014;86:11451–8.CrossRefGoogle Scholar
  23. 23.
    Gao Z, Tang D, Xu M, Chen G, Yang H. Nanoparticle-based pseudo hapten for target-responsive cargo release from a magnetic mesoporous silica nanocontainer. Chem Commun. 2014;50:6256–8.CrossRefGoogle Scholar
  24. 24.
    Zhang B, Liu B, Liao J, Chen G, Tang D. Novel electrochemical immunoassay for quantitative monitoring of biotoxin using target-responsive cargo release from mesoporous silica nanocontainers. Anal Chem. 2013;85:9245–52.CrossRefGoogle Scholar
  25. 25.
    Tang D, Liu B, Niessner R, Li P, Knopp D. Target-induced displacement reaction accompanying cargo release from magnetic mesoporous silica nanocontainers for fluorescence immunoassay. Anal Chem. 2013;85:10589–96.CrossRefGoogle Scholar
  26. 26.
    Zhuang J, Tang D, Lai W, Chen G, Yang H. Immobilization-free programmable hairpin probe for ultrasensitive electronic monitoring of nucleic acid based on a biphasic reaction mode. Anal Chem. 2014;86:8400–7.CrossRefGoogle Scholar
  27. 27.
    Han J, Sudheendra L, Kennedy I. FRET-based homogeneous immunoassay on a nanoparticle-based photonic crystal. Anal Bioanal Chem. 2015;407:5243–7.CrossRefGoogle Scholar
  28. 28.
    Yamanishi C, Chiu J, Takayama S. Systems for multiplexing homogeneous immunoassays. Bioanalysis. 2015;7:1545–56.CrossRefGoogle Scholar
  29. 29.
    Burestedt E, Nistor C, Schagerlof U, Emneus J. An enzyme flow immunoassay that uses β-galactosidase as the label and a cellobiose dehydrogenase biosensor as the label detector. Anal Chem. 2000;72:4171–7.CrossRefGoogle Scholar
  30. 30.
    Teste B, Descroix S. Colloidal nanomaterial-based immunoassay. Nanomedicine. 2012;7:917–29.CrossRefGoogle Scholar
  31. 31.
    Ren K, Wu J, Zhang Y, Yan F, Ju H. Proximity hybridization regulated DNA biogate for sensitive electrochemical immunoassay. Anal Chem. 2014;86:7494–9.CrossRefGoogle Scholar
  32. 32.
    Zhang B, Liu B, Tang D, Niessner R, Chen G, Knopp D. DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins. Anal Chem. 2012;84:5392–9.CrossRefGoogle Scholar
  33. 33.
    Yan M, Bai W, Zhu C, Huang Y, Yan J, Chen A. Design of nuclease-based target recycling signal amplification in aptasensors. Biosens Bioelectron. 2016;77:613–23.CrossRefGoogle Scholar
  34. 34.
    Liu S, Gong H, Wang Y, Wang L. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification. Biosens Bioelectron. 2016;77:818–23.CrossRefGoogle Scholar
  35. 35.
    Ding X, Wu W, Zhu Q, Zhang T, Jin W, Mu Y. Mixed-dye-based label-free and sensitive dual fluorescence for the product detection of nucleic acid isothermal multiple-self-matching-initiated amplification. Anal Chem. 2015;87:10306–14.CrossRefGoogle Scholar
  36. 36.
    Zhao Y, Chen F, Li Q, Wang L, Fan C. Isothermal amplification of nucleic acids. Chem Rev. 2015;115:12491–545.CrossRefGoogle Scholar
  37. 37.
    Zhou D, Du W, Xi Q, Ge J, Jiang J. Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection. Anal Chem. 2014;86:6763–7.CrossRefGoogle Scholar
  38. 38.
    Liu B, Chen J, Wei Q, Zhang B, Zhang L, Tang D. Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy. Biosens Bioelectron. 2015;69:241–8.CrossRefGoogle Scholar
  39. 39. RNA structure, version 5.8, University of Rochester Medical Center.

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Key Laboratory of Functional Small Organic Molecule, Ministry of Education, College of Chemistry and Chemical EngineeringJiangxi Normal UniversityNanchangChina
  2. 2.Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of ChemistryFuzhou UniversityFuzhouChina

Personalised recommendations