Microchimica Acta

, Volume 184, Issue 5, pp 1471–1479 | Cite as

Flow cytometric immunoassay for aflatoxin B1 using magnetic microspheres encoded with upconverting fluorescent nanocrystals

  • Ying Zhang
  • Zhenyu Liao
  • Yajuan Liu
  • Yajuan Wan
  • Jin Chang
  • Hanjie Wang
Original Paper


The authors describe a flow cytometric immunoassay for aflatoxin B1 (AFB1). It has three distinct features: (a) Magnetic microspheres encoded with upconverting nanocrystals (UCNMMs) are used as fluorescent labels. These have the advantage of non-overlapping spectra and lacking crosstalk between the encoding signal and reporter signal via the low-energy near-infrared (NIR) light excitation; (b) phycoerythrin-labeled secondary antibodies are used to amplify the reporter signal; (c) The use of magnetic nanoparticles facilitates the rapid separation and specific purification of the analyte (AFB1). This assay has a detection limit of 9 pg·mL−1 and a broad working range for AFB1, requires a 50 μL sample only, and can be completed within 2 h with good accuracy and high reproducibility. It is perceived that such multifluorescent UCNMMs, whose color depends on the kind of dopants (Yb, Er, Tm, Mn) in the NaYF4 host lattice, represent a promising tool for the analysis of mycotoxins and other analytes.

Graphical abstract

Schematic of the UCNMM-based indirective competitive immunoassay for AFB1 using the flow cytometric analysis (FCA) technology. The UCNMMs are prepared by doping the upconversion nanocrystals and magnetic nanoparticles inside the mesoporous polystyrene microspheres as the self-healing encapsulation strategy.


Mycotoxin Upconversion nanocrystals Magnetic nanoparticles Mesoporous polystyrene microspheres Self-healing encapsulation strategy Flow cytometric analysis Indirect competitive immunoassay 



The authors gratefully acknowledge National Natural Science Foundation of China (31401578, 51373117, 51303126, 51573128 and 81402575), Tianjin Natural Science Foundation (13JCZDJC33200 and 15JCQNJC03100).

Compliance with ethical standards

The authors declare no competing financial interests.

Supplementary material

604_2017_2116_MOESM1_ESM.docx (1002 kb)
ESM 1 (DOCX 0.97 mb)


  1. 1.
    Creppy EE (2002) Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicol Lett 127(1–3):19–28. doi: 10.1016/S0378-4274(01)00479-9 CrossRefGoogle Scholar
  2. 2.
    Hussein HS, Brasel JM (2001) Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 167(2):101–134. doi: 10.1016/S0300-483X(01)00471-1 CrossRefGoogle Scholar
  3. 3.
    Beizaei A, O’ Kane SL, Kamkar A, Misaghi A, Henehan G, Cahill DJ (2015) Highly sensitive toxin microarray assay for aflatoxin B1 detection in cereals. Food Control 57:210–215. doi: 10.1016/j.foodcont.2015.03.039 CrossRefGoogle Scholar
  4. 4.
    Min W-K, Kweon D-H, Park K, Park Y-C, Seo J-H (2011) Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli. Food Chem 126(3):1316–1323. doi: 10.1016/j.foodchem.2010.11.088 CrossRefGoogle Scholar
  5. 5.
    Guchi E (2015) Implication of aflatoxin contamination in agricultural products. American Journal of Food and Nutrition 3(1):12–20. doi: 10.12691/ajfn-3-1-3 Google Scholar
  6. 6.
    Rahmani A, Jinap S, Soleimany F (2009) Qualitative and quantitative analysis of mycotoxins. Compr Rev Food Sci Food Saf 8(3):202–251. doi: 10.1111/j.1541-4337.2009.00079.x CrossRefGoogle Scholar
  7. 7.
    Li Z, Yu Y, Li Z, Wu T (2015) A review of biosensing techniques for detection of trace carcinogen contamination in food products. Anal Bioanal Chem 407(10):2711–2726CrossRefGoogle Scholar
  8. 8.
    Rahmani A, Jinap S, Soleimany F (2010) Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin a and zearalenone in cereals using HPLC-FLD. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 27(12):1683–1693CrossRefGoogle Scholar
  9. 9.
    Zhang Z, Hu X, Zhang Q, Li P (2016) Determination for multiple mycotoxins in agricultural products using HPLC-MS/MS via a multiple antibody immunoaffinity column. J Chromatogr B Anal Technol Biomed Life Sci 1021:145–152CrossRefGoogle Scholar
  10. 10.
    He T, Wang Y, Li P, Zhang Q, Lei J, Zhang Z, Ding X, Zhou H, Zhang W (2014) Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol. Anal Chem 86(17):8873–8880CrossRefGoogle Scholar
  11. 11.
    Deng G, Xu K, Sun Y, Chen Y, Zheng T, Li J (2013) High sensitive immunoassay for multiplex mycotoxin detection with photonic crystal microsphere suspension array. Anal Chem 85(5):2833–2840CrossRefGoogle Scholar
  12. 12.
    Chauhan R, Singh J, Sachdev T, Basu T, Malhotra BD (2016) Recent advances in mycotoxins detection. Biosens Bioelectron 81:532–545CrossRefGoogle Scholar
  13. 13.
    Wang Y, Liu N, Ning B, Liu M, Lv Z, Sun Z, Peng Y, Chen C, Li J, Gao Z (2012) Simultaneous and rapid detection of six different mycotoxins using an immunochip. Biosens Bioelectron 34(1):44–50CrossRefGoogle Scholar
  14. 14.
    Malhotra BD, Srivastava S, Ali MA, Singh C (2014) Nanomaterial-based biosensors for food toxin detection. Appl Biochem Biotechnol 174(3):880–896. doi: 10.1007/s12010-014-0993-0 CrossRefGoogle Scholar
  15. 15.
    Ko J, Lee C, Choo J (2015) Highly sensitive SERS-based immunoassay of aflatoxin B1 using silica-encapsulated hollow gold nanoparticles. J Hazard Mater 285:11–17CrossRefGoogle Scholar
  16. 16.
    Lv X, Li Y, Cao W, Yan T, Li Y, Du B, Wei Q (2014) A label-free electrochemiluminescence immunosensor based on silver nanoparticle hybridized mesoporous carbon for the detection of aflatoxin B 1. Sensors Actuators B Chem 202(4):53–59CrossRefGoogle Scholar
  17. 17.
    Lu Z, Chen X, Wang Y, Zheng X, Li CM (2014) Aptamer based fluorescence recovery assay for aflatoxin B1 using a quencher system composed of quantum dots and graphene oxide. Mikrochimica Acta 182:571–578CrossRefGoogle Scholar
  18. 18.
    Lee J, Jeon CH, Ahn SJ, Ha TH (2014) Highly stable colorimetric aptamer sensors for detection of ochratoxin a through optimizing the sequence with the covalent conjugation of hemin. Analyst 139(7):1622–1627. doi: 10.1039/C3AN01639K CrossRefGoogle Scholar
  19. 19.
    Yue S, Jie X, Wei L, Bin C, Dou WD, Yi Y, Qingxia L, Jianlin L, Tiesong Z (2014) Simultaneous detection of ochratoxin a and fumonisin B1 in cereal samples using an aptamer-photonic crystal encoded suspension array. Anal Chem 86(23):11797–11802CrossRefGoogle Scholar
  20. 20.
    Bonetta L (2005) Flow cytometry smaller and better. Nat Methods 2(10):785–795CrossRefGoogle Scholar
  21. 21.
    Ren W, Liu H, Yang W, Fan Y, Yang L, Wang Y, Liu C, Li Z (2013) A cytometric bead assay for sensitive DNA detection based on enzyme-free signal amplification of hybridization chain reaction. Biosens Bioelectron 49C(22):380–386CrossRefGoogle Scholar
  22. 22.
    Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, Lowe L, Chen R, Shivraj L, Agadir A, Campos R, Ernst D, Gaur A (2004) Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clin Immunol 110(3):252–266. doi: 10.1016/j.clim.2003.11.017 CrossRefGoogle Scholar
  23. 23.
    Vignali DAA (2000) Multiplexed particle-based flow cytometric assays. J Immunol Methods 243(1–2):243–255. doi: 10.1016/S0022-1759(00)00238-6 CrossRefGoogle Scholar
  24. 24.
    Wang H-Q, Liu T-C, Cao Y-C, Huang Z-L, Wang J-H, Li X-Q, Zhao Y-D (2006) A flow cytometric assay technology based on quantum dots-encoded beads. Anal Chim Acta 580(1):18–23. doi: 10.1016/j.aca.2006.07.048 CrossRefGoogle Scholar
  25. 25.
    Sathe TR, Agrawal A, Nie S (2006) Mesoporous silica beads embedded with semiconductor quantum dots and iron oxide nanocrystals: dual-function Microcarriers for optical encoding and magnetic separation. Anal Chem 78(16):5627–5632. doi: 10.1021/ac0610309 CrossRefGoogle Scholar
  26. 26.
    Zhang Y, Dong C, Su L, Wang H, Gong X, Wang H, Liu J, Chang J (2016) Multifunctional microspheres encoded with upconverting nanocrystals and magnetic nanoparticles for rapid separation and immunoassays. ACS Appl Mater Interfaces 8(1):745–753. doi: 10.1021/acsami.5b09913 CrossRefGoogle Scholar
  27. 27.
    Gorris HH, Wolfbeis OS (2013) Photon-upconverting nanoparticles for optical encoding and multiplexing of cells, biomolecules, and microspheres. Angew Chem 52(13):3584CrossRefGoogle Scholar
  28. 28.
    Wang M, Mi C-C, Wang W-X, Liu C-H, Wu Y-F, Xu Z-R, Mao C-B, Xu S-K (2009) Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4:Yb,Er upconversion nanoparticles. ACS Nano 3(6):1580–1586. doi: 10.1021/nn900491j CrossRefGoogle Scholar
  29. 29.
    Wu S, Duan N, Wang Z, Wang H (2011) Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin a using upconversion nanoparticles as labels. Analyst 136(11):2306–2314. doi: 10.1039/C0AN00735H CrossRefGoogle Scholar
  30. 30.
    Zhang F, Shi Q, Zhang Y, Shi Y, Ding K, Zhao D, Stucky GD (2011) Fluorescence upconversion Microbarcodes for multiplexed biological detection: nucleic acid encoding. Adv Mater 23(33):3775–3779. doi: 10.1002/adma.201101868 CrossRefGoogle Scholar
  31. 31.
    Wang H, Liu Z, Wang S, Dong C, Gong X, Zhao P, Chang J (2014) MC540 and upconverting Nanocrystal Coloaded polymeric liposome for near-infrared light-triggered photodynamic therapy and cell fluorescent imaging. ACS Appl Mater Interfaces 6(5):3219–3225. doi: 10.1021/am500097f CrossRefGoogle Scholar
  32. 32.
    Tang D, Zhong Z, Niessner R, Knopp D (2009) Multifunctional magnetic bead-based electrochemical immunoassay for the detection of aflatoxin B1 in food. Analyst 134(8):1554–1560. doi: 10.1039/B902401H CrossRefGoogle Scholar
  33. 33.
    Song E, Han W, Li J, Jiang Y, Cheng D, Song Y, Zhang P, Tan W (2014) Magnetic-encoded fluorescent multifunctional Nanospheres for simultaneous multicomponent analysis. Anal Chem 86(19):9434–9442. doi: 10.1021/ac5031286 CrossRefGoogle Scholar
  34. 34.
    Guo Y, Tian J, Liang C, Zhu G, Gui W (2013) Multiplex bead-array competitive immunoassay for simultaneous detection of three pesticides in vegetables. Microchim Acta 180(5):387–395CrossRefGoogle Scholar
  35. 35.
    Wang Y, Ning B, Peng Y, Bai J, Liu M, Fan X, Sun Z, Lv Z, Zhou C, Gao Z (2013) Application of suspension array for simultaneous detection of four different mycotoxins in corn and peanut. Biosens Bioelectron 41:391–396. doi: 10.1016/j.bios.2012.08.057 CrossRefGoogle Scholar
  36. 36.
    Guo X, Wen F, Zheng N, Luo Q, Wang H, Wang H, Li S, Wang J (2014) Development of an ultrasensitive aptasensor for the detection of aflatoxin B 1. Biosens Bioelectron 56C(1):340–344CrossRefGoogle Scholar
  37. 37.
    Lu Z, Chen X, Wang Y, Zheng X, Li CM (2015) Aptamer based fluorescence recovery assay for aflatoxin B1 using a quencher system composed of quantum dots and graphene oxide. Microchim Acta 182(3–4):571–578CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2017

Authors and Affiliations

  1. 1.College of Life SciencesTianjin University, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment TechnologyTianjinPeople’s Republic of China
  2. 2.The National Center of Supervision and Inspection for Quality of FoodTianjin Product Quality Inspection Technology Research InstituteTianjinPeople’s Republic of China
  3. 3.College of Life SciencesNankai UniversityTianjinPeople’s Republic of China

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