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Development of a magnetic separation immunoassay with high sensitivity and time-saving for detecting aflatoxin B1 in agricultural crops using nanobody

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Abstract

Detection methods with high sensitivity and short assay time are urgently required for quantitative analysis of small-molecule hazardous substances in food monitoring. In this work, a new anti-aflatoxin B1 (AFB1) nanobody was screened from an immunized nanobody library, and an ultrafast one-step detection of AFB1 without immobilization and multi-step washing was developed based on magnetic separation technology and nanobody (Nb)-alkaline phosphatase (ALP) fusion protein. Compared to conventional one-step chemiluminescent enzyme-linked immunosorbent assay (CLEIA) based on Nb-ALP, it was surprising to find the sensitivity and lowest limit of detection (LOD) of this method was significantly improved about threefold and fivefold separately, and the total assay time could be reduced to 30 from 120 min. Under optimal conditions, the developed method achieved the sensitive detection of AFB1 with LOD with 0.743 pg mL−1, IC50 = 0.33 ng mL−1, the linear range was 7.23 pg mL−1 ~ 12.38 ng mL−1, and showed powerful tolerance and utility for complex matrix environments in sample detection. It is believed this method could provide a newly way for the quick and sensitive detection of AFB1 and could expand the application of Nbs.

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References

  1. Caceres I, Snini SP, Puel O, Mathieu F (2018) Streptomyces roseolus, a promising biocontrol agent against Aspergillus flavus, the main aflatoxin B1 producer. Toxins 10:442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Battilani P, Toscano P, Van der Fels-Klerx HJ, Moretti A, Camardo Leggieri M, Brera C, Rortais A, Goumperis T, Robinson T (2016) Aflatoxin B1 contamination in maize in Europe increases due to climate change. Sci Rep 6:24328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Streit E, Schatzmayr G, Tassis P, Tzika E, Marin D, Taranu I, Tabuc C, Nicolau A, Aprodu I, Puel O, Oswald IP (2012) Current situation of mycotoxin contamination and co-occurrence in animal feed–focus on Europe. Toxins (Basel) 4:788–809

    Article  CAS  PubMed  Google Scholar 

  4. Cimbalo A, Alonso-Garrido M, Font G, Manyes L (2020) Toxicity of mycotoxins in vivo on vertebrate organisms: a review. Food Chem Toxicol 137:111161

    Article  CAS  PubMed  Google Scholar 

  5. Mahato DK, Lee KE, Kamle M, Devi S, Dewangan KN, Kumar P, Kang SG (2019) Aflatoxins in food and feed: an overview on prevalence detection and control strategies. Front Microbiol 10:2266

    Article  PubMed  PubMed Central  Google Scholar 

  6. Abrar M, Anjum FM, Butt MS, Pasha I, Randhawa MA, Saeed F, Waqas K (2013) Aflatoxins: biosynthesis, occurrence, toxicity, and remedies. Crit Rev Food Sci Nutr 53:862–874

    Article  CAS  PubMed  Google Scholar 

  7. Khayoon WS, Saad B, Lee TP, Salleh B (2012) High performance liquid chromatographic determination of aflatoxins in chilli, peanut and rice using silica based monolithic column. Food Chem 133:489–496

    Article  CAS  PubMed  Google Scholar 

  8. Romera D, Mateo EM, Mateo-Castro R, Gómez JV, Gimeno-Adelantado JV, Jiménez M (2018) Determination of multiple mycotoxins in feedstuffs by combined use of UPLC-MS/MS and UPLC-QTOF-MS. Food Chem 267:140–148

    Article  CAS  PubMed  Google Scholar 

  9. Chen F, Luan C, Wang L, Wang S, Shao L (2017) Simultaneous determination of six mycotoxins in peanut by high-performance liquid chromatography with a fluorescence detector. J Sci Food Agric 97:1805–1810

    Article  CAS  PubMed  Google Scholar 

  10. Wang X, Niessner R, Tang D, Knopp D (2016) Nanoparticle-based immunosensors and immunoassays for aflatoxins. Anal Chim Acta 912:10–23

    Article  CAS  PubMed  Google Scholar 

  11. Zhang D, Li P, Zhang Q, Zhang W (2011) Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts. Biosens Bioelectron 26:2877–2882

    Article  CAS  PubMed  Google Scholar 

  12. Gazzaz SS, Rasco BA, Dong FM (1992) Application of immunochemical assays to food analysis. Crit Rev Food Sci Nutr 32:197–229

    Article  CAS  PubMed  Google Scholar 

  13. Du J, Chen X, Liu K, Zhao D, Bai Y (2022) Dual recognition and highly sensitive detection of Listeria monocytogenes in food by fluorescence enhancement effect based on Fe3O4@ZIF-8-aptamer. Sens Actuators B 360:131654

    Article  CAS  Google Scholar 

  14. Zhang W, Serpe MJ (2017) Antigen detection using fluorophore-modified antibodies and magnetic microparticles. Sens Actuators B 238:441–446

    Article  CAS  Google Scholar 

  15. Yue Q, Li X, Fang J, Li M, Zhang J, Zhao G, Cao W, Wei Q (2022) Oxygen Free Radical Scavenger PtPd@PDA as a dual-mode quencher of electrochemiluminescence immunosensor for the detection of AFB1. Anal Chem 94:11476–11482

    Article  CAS  PubMed  Google Scholar 

  16. Gonzalez-Sapienza G, Rossotti MA, Tabares-da Rosa S (2017) Single-domain antibodies as versatile affinity reagents for analytical and diagnostic applications. Front Immunol. https://doi.org/10.3389/fimmu.2017.00977

    Article  PubMed  PubMed Central  Google Scholar 

  17. Shu M, Xu Y, Wang D, Liu X, Li Y, He Q, Tu Z, Qiu Y, Ji Y, Wang X (2015) Anti-idiotypic nanobody: a strategy for development of sensitive and green immunoassay for Fumonisin B1. Talanta 143:388–393

    Article  CAS  PubMed  Google Scholar 

  18. Tang Z, Wang X, Lv J, Hu X, Liu X (2018) One-step detection of ochratoxin A in cereal by dot immunoassay using a nanobody-alkaline phosphatase fusion protein. Food Control 92:430–436

    Article  CAS  Google Scholar 

  19. Ren W, Li Z, Xu Y, Wan D, Barnych B, Li Y, Tu Z, He Q, Fu J, Hammock BD (2019) One-step ultrasensitive bioluminescent enzyme immunoassay based on nanobody/nanoluciferase fusion for detection of aflatoxin B1 in cereal. J Agric Food Chem 67:5221–5229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dunlop EH, Feiler WA, Mattione MJ (1984) Magnetic separation in biotechnology. Biotechnol Adv 2:63–74

    Article  CAS  PubMed  Google Scholar 

  21. Wang F, Li ZF, Yang YY, Wan DB, Vasylieva N, Zhang YQ, Cai J, Wang H, Shen YD, Xu ZL, Hammock BD (2020) Chemiluminescent enzyme immunoassay and bioluminescent enzyme immunoassay for tenuazonic acid mycotoxin by exploitation of nanobody and nanobody-nanoluciferase fusion. Anal Chem 92:11935–11942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Di S, Ning T, Yu J, Chen P, Yu H, Wang J, Yang H, Zhu S (2020) Recent advances and applications of magnetic nanomaterials in environmental sample analysis. TrAC Trends Anal Chem 126:115864

    Article  CAS  Google Scholar 

  23. Zhao F, Shi R, Liu R, Tian Y, Yang Z (2021) Application of phage-display developed antibody and antigen substitutes in immunoassays for small molecule contaminants analysis: a mini-review. Food Chem 339:128084

    Article  CAS  PubMed  Google Scholar 

  24. Sun T, Zhao Z, Liu W, Xu Z, He H, Ning B, Jiang Y, Gao Z (2020) Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B nanobody-alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B. Anal Chim Acta 1108:28–36

    Article  CAS  PubMed  Google Scholar 

  25. Zhang YY, Li LH, Wang Y, Wang H, Xu ZL, Tian YX, Sun YM, Yang JY, Shen YD (2022) Ultrasensitive and rapid colorimetric detection of paraquat via a high specific VHH nanobody. Biosens Bioelectron 205:114089

    Article  CAS  PubMed  Google Scholar 

  26. Güttler T, Aksu M, Dickmanns A, Stegmann KM, Gregor K, Rees R, Taxer W, Rymarenko O, Schünemann J, Dienemann C, Gunkel P, Mussil B, Krull J, Teichmann U, Groß U, Cordes VC, Dobbelstein M, Görlich D (2021) Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies. Embo j 40:e107985

    Article  PubMed  PubMed Central  Google Scholar 

  27. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

    Article  CAS  PubMed  Google Scholar 

  28. Mateo C, Grazu V, Palomo JM, Lopez-Gallego F, Fernandez-Lafuente R, Guisan JM (2007) Immobilization of enzymes on heterofunctional epoxy supports. Nat Protoc 2:1022–1033

    Article  CAS  PubMed  Google Scholar 

  29. Hehir MJ, Murphy JE, Kantrowitz ER (2000) Characterization of heterodimeric alkaline phosphatases from Escherichia coli: an investigation of intragenic complementation. J Mol Biol 304:645–656

    Article  CAS  PubMed  Google Scholar 

  30. Sowadski JM, Handschumacher MD, Murthy HM, Foster BA, Wyckoff HW (1985) Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution. J Mol Biol 186:417–433

    Article  CAS  PubMed  Google Scholar 

  31. Kunz P, Zinner K, Mücke N, Bartoschik T, Muyldermans S, Hoheisel JD (2018) The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep 8:7934

    Article  PubMed  PubMed Central  Google Scholar 

  32. Le Basle Y, Chennell P, Tokhadze N, Astier A, Sautou V (2020) Physicochemical stability of monoclonal antibodies: a review. J Pharm Sci 109:169–190

    Article  PubMed  Google Scholar 

  33. Goldman ER, Liu JL, Zabetakis D, Anderson GP (2017) Enhancing stability of camelid and shark single domain antibodies: an overview. Front Immunol 8:865

    Article  PubMed  PubMed Central  Google Scholar 

  34. Tang X, Li P, Zhang Q, Zhang Z, Zhang W, Jiang J (2017) Time-resolved fluorescence immunochromatographic assay developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin and zearalenone in maize and its products. Anal Chem 89:11520–11528

    Article  CAS  PubMed  Google Scholar 

  35. Yan T, Zhu J, Li Y, He T, Yang Y, Liu M (2022) Development of a biotinylated nanobody for sensitive detection of aflatoxin B(1) in cereal via ELISA. Talanta 239:123125

    Article  CAS  PubMed  Google Scholar 

  36. 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 86:8873–8880

    Article  CAS  PubMed  Google Scholar 

  37. Zhang X, Liao X, Wu Y, Xiong W, Du J, Tu Z, Yang W, Wang D (2022) A sensitive electrochemical immunosensing interface for label-free detection of aflatoxin B(1) by attachment of nanobody to MWCNTs-COOH@black phosphorene. Anal Bioanal Chem 414:1129–1139

    Article  CAS  PubMed  Google Scholar 

  38. Liu X, Wen Y, Wang W, Zhao Z, Han Y, Tang K, Wang D (2020) Nanobody-based electrochemical competitive immunosensor for the detection of AFB(1) through AFB(1)-HCR as signal amplifier. Mikrochim Acta 187:352

    Article  CAS  PubMed  Google Scholar 

  39. Xu W, Xiong Y, Lai W, Xu Y, Li C, Xie M (2014) A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots. Biosens Bioelectron 56:144–150

    Article  CAS  PubMed  Google Scholar 

  40. Li J, Zhao X, Chen LJ, Qian HL, Wang WL, Yang C, Yan XP (2019) p-Bromophenol-enhanced bienzymatic chemiluminescence competitive immunoassay for ultrasensitive determination of aflatoxin B(1). Anal Chem 91:13191–13197

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This research has received funding support from (20SWAQK16, 2019GGRC03, 2019CXTD03), Heilongjiang Province Key Laboratory of Microecology-Immune Regulation Network and Related Diseases (2021-SZD-JC-005), 2021 Heilongjiang Provincial Health and Wellness Committee Project (20210202040058)

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Authors and Affiliations

  1. Department of Immunology, School of Basic Medicine, Jiamusi University, Jiamusi , Heilongjiang, China

    Xinyang Wang, Weili Shen & Changli Shao

  2. Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, China

    Xinyang Wang, Hu Zuo, Weili Shen, Yiyang Zhang, Ruonan Liu, Lu Geng, Wen Wang & Tieqiang Sun

  3. Key Laboratory of Microecology-Immune Regulatory Network and Related Diseases, School of Basic Medicine, Jiamusi University, Jiamusi, Heilongjiang Province, China

    Changli Shao

  4. School of Information Science and Engineering, Xinjiang University, Xinjiang, China

    Wentao Liu

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  1. Xinyang Wang
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Contributions

XW: conceptualization, writing—original draft, methodology, and writing—review and editing. WL: methodology, investigation, and software. HZ: investigation and methodology. WS: investigation and methodology. YZ: methodology and software. RL: methodology and validation. LG: investigation and validation. WW: investigation and validation. CS: writing—review and editing, supervision, and methodology. TS: investigation, writing—review and editing, validation, and funding acquisition.

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Correspondence to Changli Shao or Tieqiang Sun.

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Wang, X., Liu, W., Zuo, H. et al. Development of a magnetic separation immunoassay with high sensitivity and time-saving for detecting aflatoxin B1 in agricultural crops using nanobody. Eur Food Res Technol 249, 1125–1136 (2023). https://doi.org/10.1007/s00217-023-04202-3

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