Analytical and Bioanalytical Chemistry

, Volume 411, Issue 11, pp 2405–2414 | Cite as

Exonuclease I-assisted fluorescent method for ochratoxin A detection using iron-doped porous carbon, nitrogen-doped graphene quantum dots, and double magnetic separation

  • Chengke WangEmail author
  • Rong Tan
  • Jiangyu Li
  • Zexiang Zhang
Research Paper


In this paper, a fluorescent method was developed for ochratoxin A (OTA) detection that uses iron-doped porous carbon (MPC) and aptamer-functionalized nitrogen-doped graphene quantum dots (NGQDs-Apt) as probes. In this method, the adsorbance of the NGQDs-Apt on the MPC due to a π–π interaction between the aptamer and the MPC results in the quenching of the fluorescence of the NGQDs-Apt. However, since OTA interacts strongly with the aptamer, the presence of OTA leads to the detachment of the NGQDs-Apt from the MPC, resulting in the resumption of fluorescence from the NGQDs-Apt. When exonuclease I (Exo I) is also added to the solution, this exonuclease specifically digests the aptamer, leading to the release of the OTA back into the solution. This free OTA then interacts with another MPC–NGQDs-Apt system, inducing the release of more NGQDs into the solution, which enhances the fluorescent intensity compared to that of the system with no Exo I. Utilizing this behavior of OTA in the presence of NGQDs-Apt, it was possible to detect concentrations of OTA ranging from 10 to 5000 nM, with a limit of detection of 2.28 nM. Our method was tested by applying it to the detection of OTA in wheat and corn samples. This method has four advantages: (1) the magnetic porous carbon is easy to prepare, its porosity enhances its loading capacity for NGQDs, it highly efficiently quenches the fluorescence of the NGQDs, and its magnetic properties facilitate the separation of the MPC from other species in solution; (2) applying double magnetic separation decreases the background signal; (3) Exo I digests the free aptamer effectively, which allows the resulting free OTA to induce the release of more NGQDs-Apt, ultimately enhancing the fluorescent signal; and (4) the proposed method presented high sensitivity and a wide linear detection range. This method may prove helpful in food safety analysis and new biosensor development (achieved by using different aptamer sequences to that used in the present work).

Graphical abstract

Exonuclease I (Exo I)-assisted fluorescent method for ochratoxin A (OTA) detection using magnetic porous carbon (MPC), nitrogen-doped graphene quantum dots (NGQDs), and double magnetic separation


Ochratoxin A Porous carbon Magnetic separation Exonuclease I Graphene quantum dots 



This study was funded by the National Natural Science Foundation of China (no. 21305032), the China Postdoctoral Science Foundation (no. 2014 M551522), Jiangsu Planned Projects for Postdoctoral Research Funds (no. 1402073B), and Hong Kong Scholar Program (no. XJ2017008).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1684_MOESM1_ESM.pdf (355 kb)
ESM 1 (PDF 262 kb)


  1. 1.
    Ostry V, Malir F, Toman J, Grosse Y. Mycotoxins as human carcinogens—the IARC monographs classification. Mycotoxin Res. 2017;33(1):65–73.Google Scholar
  2. 2.
    Liew WPP, Mohd-Redzwan S. Mycotoxin: its impact on gut health and microbiota. Front Cell Infect Microbiol. 2018;8:60.CrossRefGoogle Scholar
  3. 3.
    Lee HJ, Ryu D. Worldwide occurrence of mycotoxins in cereals and cereal-derived food products: public health perspectives of their co-occurrence. J Agric Food Chem. 2017;65(33):7034–51.CrossRefGoogle Scholar
  4. 4.
    Zhang YY, Pei F, Fang Y, Li P, Zhao Y, Shen F, et al. Comparison of concentration and health risks of 9 fusarium mycotoxins in commercial whole wheat flour and refined wheat flour by multi-IAC-HPLC. Food Chem. 2019;275:763–9.CrossRefGoogle Scholar
  5. 5.
    Tang ZW, Wang XR, Lv JW, Hu XR, Liu X. One-step detection of ochratoxin a in cereal by dot immunoassay using a nanobody-alkaline phosphatase fusion protein. Food Control. 2018;92:430–6.CrossRefGoogle Scholar
  6. 6.
    Sun ZC, Lv JW, Liu X, Tang ZW, Wang XR, Xu Y, et al. Development of a nanobody-AviTag fusion protein and its application in a streptavidin-biotin-amplified enzyme-linked immunosorbent assay for ochratoxin A in cereal. Anal Chem. 2018;90(17):10628–34.Google Scholar
  7. 7.
    Majdinasab M, Zareian M, Zhang Q, Li PW. Development of a new format of competitive immunochromatographic assay using secondary antibody-europium nanoparticle conjugates for ultrasensitive and quantitative determination of ochratoxin A. Food Chem. 2019;275:721–9.Google Scholar
  8. 8.
    Jalalvand AR. Fabrication of a novel and high-performance amperometric sensor for highly sensitive determination of ochratoxin A in juice samples. Talanta. 2018;188:225–31.Google Scholar
  9. 9.
    Majdinasab M, Hayat A, Marty JL. Aptamer-based assays and aptasensors for detection of pathogenic bacteria in food samples. TRAC—Trend Anal Chem. 2018;107:60–77.Google Scholar
  10. 10.
    Farzin L, Shamsipur M, Sheibani S. A review: aptamer-based analytical strategies using the nanomaterials for environmental and human monitoring of toxic heavy metals. Talanta. 2017;174:619–27.CrossRefGoogle Scholar
  11. 11.
    Deng B, Lin YW, Wang C, Li F, Wang ZX, Zhang HQ, et al. Aptamer binding assays for proteins: the thrombin example—a review. Anal Chim Acta. 2014;837:1–15.Google Scholar
  12. 12.
    Cunha I, Biltes R, Sales MGF, Vasconcelos V. Aptamer-based biosensors to detect aquatic phycotoxins and cyanotoxins. Sensors. 2018;18(7):2367.CrossRefGoogle Scholar
  13. 13.
    Zhou W, Huang PJ, Ding J, Liu J. Aptamer-based biosensors for biomedical diagnostics. Analyst. 2014;139(11):2627–40.CrossRefGoogle Scholar
  14. 14.
    Mok W, Li YF. Recent progress in nucleic acid aptamer-based biosensors and bioassays. Sensors. 2008;8(11):7050–84.CrossRefGoogle Scholar
  15. 15.
    Amaya-Gonzalez S, de los Santos Alvarez N, Miranda-Ordieres AJ, Lobo-Castanon MJ. Aptamer-based analysis: a promising alternative for food safety control. Sensors. 2013;13(12):16292–311.CrossRefGoogle Scholar
  16. 16.
    Wang CK, Tan R, Chen D. Fluorescence method for quickly detecting ochratoxin A in flour and beer using nitrogen doped carbon dots and silver nanoparticles. Talanta. 2018;182:363–70.Google Scholar
  17. 17.
    Wang CK, Chen D, Wang QQ, Wang QX. Aptamer-based resonance light scattering for sensitive detection of acetamiprid. Anal Sci. 2016;32(7):757–62.CrossRefGoogle Scholar
  18. 18.
    Wu YQ, Qiu XC, Liang F, Zhang QK, Koo A, Dai YN, et al. A metal-organic framework-derived bifunctional catalyst for hybrid sodium-air batteries. Appl Catal B—Environ. 2019;241:407–14.Google Scholar
  19. 19.
    Nguyen TN, Ebrahim FM, Stylianou KC. Photoluminescent, upconversion luminescent and nonlinear optical metal-organic frameworks: from fundamental photophysics to potential applications. Coord Chem Rev. 2018;377:259–306.CrossRefGoogle Scholar
  20. 20.
    Gupta V, Tyagi S, Paul AK. Development of biocompatible iron–carboxylate metal organic frameworks for pH-responsive drug delivery application. J Nanosci Nanotechnol. 2019;19(2):646–54.Google Scholar
  21. 21.
    Gao XY, Zhu G, Zhang XJ, Hu T. Porous carbon materials derived from in situ construction of metal-organic frameworks for high-performance sodium ions batteries. Microporous Mesoporous Mater. 2019;273:156–62.CrossRefGoogle Scholar
  22. 22.
    Nadar SS, Rathod VK. Magnetic-metal organic framework (magnetic-MOF): a novel platform for enzyme immobilization and nanozyme applications. Int J Biol Macromol. 2018;120:2293–302.CrossRefGoogle Scholar
  23. 23.
    Ajoyan Z, Marino P, Howarth AJ. Green applications of metal-organic frameworks. CrystEngComm. 2018;20(39):5899–912.CrossRefGoogle Scholar
  24. 24.
    Zhu SY, Yan B. A novel sensitive fluorescent probe of S2O8 2− and Fe3+ based on covalent post-functionalization of a zirconium(IV) metal-organic framework. Dalton Trans. 2018;47(33):11586–92.CrossRefGoogle Scholar
  25. 25.
    Xu PP, Liao GF. A novel fluorescent biosensor for adenosine triphosphate detection based on a metal-organic framework coating polydopamine layer. Materials. 2018;11(9):1616.CrossRefGoogle Scholar
  26. 26.
    Yu FQ, Zhou H, Shen Q. Modification of cobalt-containing MOF-derived mesoporous carbon as an effective sulfur-loading host for rechargeable lithium-sulfur batteries. J Alloys Compd. 2019;772:843–51.CrossRefGoogle Scholar
  27. 27.
    Wu ZL, Sun LP, Zhou Z, Li Q, Huo LH, Zhao H. Efficient nonenzymatic H2O2 biosensor based on ZIF-67 MOF derived co nanoparticles embedded N-doped mesoporous carbon composites. Sensors Actuators B Chem. 2018;276:142–9.CrossRefGoogle Scholar
  28. 28.
    Kang JS, Kang J, Chung DY, Son YJ, Kim S, Kim S, et al. Tailoring the porosity of MOF-derived N-doped carbon electrocatalysts for highly efficient solar energy conversion. J Mater Chem A. 2018;6(41):20170–83.CrossRefGoogle Scholar
  29. 29.
    Li BY, Chrzanowski M, Zhang YM, Ma SQ. Applications of metal-organic frameworks featuring multi-functional sites. Coord Chem Rev. 2016;307:106–29.CrossRefGoogle Scholar
  30. 30.
    Liu DX, Zou DT, Zhu HL, Zhang JY. Mesoporous metal–organic frameworks: synthetic strategies and emerging applications. Small. 2018;14(37):1801454.CrossRefGoogle Scholar
  31. 31.
    Tan HL, Tang GE, Wang ZX, Li Q, Gao J, Wu SM. Magnetic porous carbon nanocomposites derived from metal-organic frameworks as a sensing platform for DNA fluorescent detection. Anal Chim Acta. 2016;940:136–42.CrossRefGoogle Scholar
  32. 32.
    Zhu ZM, Lin XY, Wu LN, Zhao CF, Zheng YJ, Liu AL, et al. “Switch-on” fluorescent nanosensor based on nitrogen-doped carbon dots-MnO2 nanocomposites for probing the activity of acid phosphatase. Sensors Actuators B Chem. 2018;274:609–15.Google Scholar
  33. 33.
    Ganganboina AB, Doong RA. Functionalized N-doped graphene quantum dots for electrochemical determination of cholesterol through host-guest inclusion. Microchim Acta. 2018;185(11):526.CrossRefGoogle Scholar
  34. 34.
    Hai X, Feng J, Chen XW, Wang JH. Tuning the optical properties of graphene quantum dots for biosensing and bioimaging. J Mater Chem B. 2018;6(20):3219–34.CrossRefGoogle Scholar
  35. 35.
    Gao J, Zhu MM, Huang H, Liu Y, Kang ZH. Advances, challenges and promises of carbon dots. Inorg Chem Front. 2017;4(12):1963–86.CrossRefGoogle Scholar
  36. 36.
    Lin CY, Zheng HX, Huang YY, Chen ZL, Luo F, Wang J, et al. Homogeneous electrochemical aptasensor for mucin 1 detection based on exonuclease I-assisted target recycling amplification strategy. Biosens Bioelectron. 2018;117:474–9.CrossRefGoogle Scholar
  37. 37.
    Chen XX, Hong F, Cao YT, Hu FT, Wu YX, Wu DZ, et al. A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification. Talanta. 2018;189:494–501.CrossRefGoogle Scholar
  38. 38.
    Lee HJ, Cho W, Lim E, Oh M. One-pot synthesis of magnetic particle-embedded porous carbon composites from metal-organic frameworks and their sorption properties. Chem Commun. 2014;50(41):5476–9.CrossRefGoogle Scholar
  39. 39.
    Wang C, Dong X, Liu Q, Wang K. Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction. Anal Chim Acta. 2015;860:83–8.Google Scholar
  40. 40.
    Lv L, Li D, Liu R, Cui C, Guo Z. Label-free aptasensor for ochratoxin A detection using SYBR gold as a probe. Sensors Actuators B Chem. 2017;246:647–52.Google Scholar
  41. 41.
    Zhang JJ, Sun Y, Lu JZ. A novel bioluminescent detection of exonuclease I activity based on terminal deoxynucleotidyl transferase-mediated pyrophosphate release. Luminescence. 2018;33(7):1157–63.CrossRefGoogle Scholar
  42. 42.
    Pu Y, Zhu Z, Han D, Liu HX, Liu J, Liao J, et al. Insulin-binding aptamer-conjugated graphene oxide for insulin detection. Analyst. 2011;136(20):4138–40.CrossRefGoogle Scholar
  43. 43.
    Wu YR, Phillips JA, Liu HP, Yang RH, Tan WH. Carbon nanotubes protect DNA strands during cellular delivery. ACS Nano. 2008;2(10):2023–8.CrossRefGoogle Scholar
  44. 44.
    Ma Y, Ji G, Lee JY. Synthesis of mixed-conducting carbon coated porous γ-Fe2O3 microparticles and their properties for reversible lithium ion storage. J Mater Chem. 2011;21(34):13009–14.CrossRefGoogle Scholar
  45. 45.
    Lin-Vien D, Colthup NB, Fateley WG, Grasselli JG. Chapter 10: Compounds containing –NH2, –NHR, and –NR2 groups. The handbook of infrared and Raman characteristic frequencies of organic molecules. San Diego: Academic Press; 1991. p. 155–78.Google Scholar
  46. 46.
    Yang C, Wang Y, Marty JL, Yang XR. Aptamer-based colorimetric biosensing of ochratoxin A using unmodified gold nanoparticles indicator. Biosens Bioelectron. 2011;26(5):2724–7.Google Scholar
  47. 47.
    Radi A-E, Muñoz-Berbel X, Cortina-Puig M, Marty J-L. An electrochemical immunosensor for ochratoxin A based on immobilization of antibodies on diazonium-functionalized gold electrode. Electrochim Acta. 2009;54(8):2180–4.Google Scholar
  48. 48.
    Lv L, Cui C, Liang C, Quan W, Wang S, Guo Z. Aptamer-based single-walled carbon nanohorn sensors for ochratoxin A detection. Food Control. 2016;60:296–301.Google Scholar
  49. 49.
    Wang R, Xiang Y, Zhou X, Liu LH, Shi H. A reusable aptamer-based evanescent wave all-fiber biosensor for highly sensitive detection of ochratoxin A. Biosens Bioelectron. 2015;66:11–8.Google Scholar
  50. 50.
    Sheng L, Ren J, Miao Y, Wang J, Wang E. PVP-coated graphene oxide for selective determination of ochratoxin A via quenching fluorescence of free aptamer. Biosens Bioelectron. 2011;26(8):3494–9.Google Scholar
  51. 51.
    Bueno D, Muñoz R, Marty JL. Fluorescence analyzer based on smartphone camera and wireless for detection of ochratoxin A. Sensors Actuators B Chem. 2016;232:462–8.Google Scholar

Copyright information

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

Authors and Affiliations

  • Chengke Wang
    • 1
    Email author
  • Rong Tan
    • 1
  • Jiangyu Li
    • 1
  • Zexiang Zhang
    • 1
  1. 1.College of Food and Biological EngineeringJiangsu UniversityZhenjiangChina

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