Development and Application of an Optical Biosensor Immunoassay for Aflatoxin M1 in Bovine Milk

  • Harvey E. IndykEmail author
  • Sowmya Chetikam
  • Brendon D. Gill
  • Jackie E. Wood
  • David C. Woollard


An automated optical biosensor-based immunoassay exploiting surface plasmon resonance detection for the quantitation of aflatoxin M1 (AFM1) in milk and milk powders is described. A monoclonal antibody and an immobilized protein–AFM1 conjugate are utilized in a simple inhibition format following aqueous extraction and immunoaffinity clean-up of the sample, thereby avoiding the need for signal amplification techniques. The sensor surface is stable over multiple regeneration cycles, and the technique yields a method detection limit of 0.1 ng g−1, which is five times lower than the European Commission maximum residue limit. The described antibody-based biosensor technique provides the advantages of quantitative data, automation, and real-time and non-labeled detection of AFM1. The method therefore facilitates routine quantitative threshold-level screening for the identification of potential non-compliance of AFM1 content prior to confirmatory analysis by reference chromatographic methods and may be considered to complement the enzyme-linked immunosorbent assay technique.


Aflatoxin M1 Milk Biosensor Surface plasmon resonance Immunoassay 



The authors acknowledge Dr. Terry Cooney (Analytica Laboratories Ltd., Hamilton, New Zealand) for LC–tandem mass spectrometry analyses and the support and encouragement of Dr. Robert Crawford and Fonterra Co-operative Group Ltd. throughout this study.

Compliance with Ethical Standards

Conflict of Interest

Harvey Indyk declares that he has no conflict of interest. Sowmya Chetikam declares that she has no conflict of interest. Brendon Gill declares that he has no conflict of interest. Jackie Wood declares that she has no conflict of interest. David Woollard declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Not applicable.


  1. AOAC (2005) AOAC Official Method 2000.08. Aflatoxin M1 in liquid milk. Immunoaffinity column by liquid chromatography. In: Official methods of analysis of AOAC International, 18th edn. AOAC, Gaithersburg, MDGoogle Scholar
  2. Armbruster DA, Pry T (2008) Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 29(Suppl 1):S49–S52PubMedPubMedCentralGoogle Scholar
  3. Benkerroum N (2016) Mycotoxins in dairy products: a review. Int Dairy J 62:63–75. CrossRefGoogle Scholar
  4. Campagnollo FB, Ganev KC, Khaneghah AM, Portela JB, Cruz AG, Granato D, Corassin CH, Oliveira CAF, Sant'Ana AS (2016) The occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin M1: a review. Food Control 68:310–329. CrossRefGoogle Scholar
  5. Daly SJ, Keating GJ, Dillon PP, Manning BM, O'Kennedy R, Lee HA, Morgan MRA (2000) Development of surface plasmon resonance-based immunoassy for aflatoxin B1. J Agric Food Chem 48:5097–5104. CrossRefPubMedGoogle Scholar
  6. EU Commission (2014) Commission Regulation (EU) No 519/2014 of 16 May 2014 amending Regulation (EC) No 401/2006 as regards methods of sampling of large lots, spices and food supplements, performance criteria for T-2, HT-2 toxin and citrinin and screening methods of analysis. Off J Eur Union L147:29–43Google Scholar
  7. Goode JA, Rushworth JVH, Millner PA (2015) Biosensor regeneration: a review of common techniques and outcomes. Langmuir 31:6267–6276. CrossRefPubMedGoogle Scholar
  8. Guider R, Gandolfi D, Chalyan T, Pasquardini L, Samusenko A, Pucker G, Pederzolli C, Pavesi L (2015) Design and optimization of SiON ring resonator-based biosensors for aflatoxin M1 detection. Sensors 15:17300–17312. CrossRefPubMedGoogle Scholar
  9. Horwitz W, Albert R (2006) The Horwitz ratio (HorRat): a useful index of method performance with respect to precision. J AOAC Int 89:1095–1109PubMedGoogle Scholar
  10. Iqbal SZ, Jinap S, Pirouz AA, Faizal AR (2015) Aflatoxin M1 in milk and dairy products, occurrence and recent challenges: a review. Trends Food Sci Technol 46:110–119. CrossRefGoogle Scholar
  11. Karczmarczyk A, Dubiak-Szepietowska M, Vorobii M, Rodriguez-Emmenegger C, Dostálek J, Feller K-H (2016) Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes. Biosens Bioelectron 81:159–165. CrossRefPubMedGoogle Scholar
  12. Ketney O, Santini A, Oancea S (2017) Recent aflatoxin survey data in milk and milk products: a review. Int J Dairy Technol 70:320–331. CrossRefGoogle Scholar
  13. Klingelhöfer D, Zhu Y, Braun M, Bendels MHK, Brüggmann D, Groneberg DA (2018) Aflatoxin—publication analysis of a global health threat. Food Control 89:280–290. CrossRefGoogle Scholar
  14. Li W, Powers S, Dai SY (2014) Using commercial immunoassay kits for mycotoxins: “joys and sorrows”. World Mycotoxin J 7:417–430. CrossRefGoogle Scholar
  15. Maragos CM (2004) Emerging technologies for mycotoxin detection. J Toxicol Toxin Rev 23:317–344. CrossRefGoogle Scholar
  16. Maragos CM (2016) Multiplexed biosensors for mycotoxins. J AOAC Int 99:849–860. CrossRefPubMedGoogle Scholar
  17. McGrath TF, Elliott CT, Fodey TL (2012) Biosensors for the analysis of microbiological and chemical contaminants in food. Anal Bioanal Chem 403:75–92. CrossRefPubMedGoogle Scholar
  18. Mohammadi H (2011) A review of aflatoxin M1, milk, and milk products. In: Guevara-Gonzalez RG (ed) aflatoxins – biochemistry and molecular biology. InTech, London, pp 397–414 Available from: Accessed 22 Feb 2019Google Scholar
  19. Shephard GS (2016) Current status of mycotoxin analysis: a critical review. J AOAC Int 99:842–848. CrossRefPubMedGoogle Scholar
  20. Situ C, Wylie ARG, Douglas A, Elliott CT (2008) Reduction of severe bovine serum associated matrix effects on carboxymethylated dextran coated biosensor surfaces. Talanta 76:832–836. CrossRefPubMedGoogle Scholar
  21. Su GCC (1998) A comparison of statistical and empirical detection limits. J AOAC Int 81:105–110Google Scholar
  22. Thompson CS, Traynor IM, Fodey TL, Faulkner DV, Crooks SRH (2017) Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor. Talanta 172:120–125. CrossRefPubMedGoogle Scholar
  23. Vaisocherová H, Brynda E, Homola J (2015) Functionalizable low-fouling coatings for label-free biosensing in complex biological media: advances and applications. Anal Bioanal Chem 407:3927–3953. CrossRefPubMedGoogle Scholar
  24. van der Gaag B, Spath S, Dietrich H, Stigter E, Boonzaaijer G, van Osenbruggen T, Koopal K (2003) Biosensors and multiple mycotoxin analysis. Food Control 14:251–254. CrossRefGoogle Scholar
  25. Vidal JC, Bonel L, Ezquerra A, Hernández S, Bertolín JR, Cubel C, Castillo JR (2013) Electrochemical affinity biosensors for detection of mycotoxins: a review. Biosens Bioelectron 49:146–158. CrossRefPubMedGoogle Scholar
  26. Visentin J, Couzi L, Dromer C, Neau-Cransac M, Guidicelli G, Veniard V, Coniat KN, Merville P, Di Primo C, Taupin J-L (2018) Overcoming non-specific binding to measure the active concentration and kinetics of serum anti-HLA antibodies by surface plasmon resonance. Biosens Bioelectron 117:191–200. CrossRefPubMedGoogle Scholar
  27. Wang Y, Dostálek J, Knoll W (2009) Long range surface plasmon-enhanced fluorescence spectroscopy for the detection of aflatoxin M1 in milk. Biosens Bioelectron 24:2264–2267. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Fonterra Co-operative Group LtdWaitoaNew Zealand
  2. 2.Hill LaboratoriesHamiltonNew Zealand

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