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Nanobody-based electrochemical competitive immunosensor for the detection of AFB1 through AFB1-HCR as signal amplifier

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Abstract

A novel nanobody (Nb)-based voltammetric immunosensor coupled with horseradish peroxidase concatemer–modified hybridization chain reaction (HRP-HCR) signal amplifying system is described to realize the rapid and ultrasensitive detection of AFB1. To design such an immunoassay, anti-AFB1 Nbs with smaller molecular size were coated densely onto the surface of Au nanoparticle-tungsten disulfide-multi-walled carbon nanotubes (AuNPs/WS2/MWCNTs) functional nanocomposites as an effective molecular recognition element, whereas AFB1-streptavidin (AFB1-SA) conjugates were ingeniously bound with biotinylated HCR dsDNA nanostructures as the competitor, amplifier, and signal report element. In the presence of AFB1 targets, a competitive immunoreaction was performed between the analyte and AFB1-SA-labeled HCR (AFB1-HCR) platform. Upon the addition of SA-modified polyHRP (SA-polyHRP), AFB1-HCR nanostructures containing abundant biotins were allowed to cross-link to a quantity of HRP by streptavidin−biotin chemistry for signal amplification and signal conversion. Under optimal conditions, the immunosensor displayed a good linear correlation toward AFB1 ranging from 0.5 to 10 ng mL−1 with a sensitivity of 2.7 μA • (mL ng−1) and an ultralow limit of detection (LOD) of 68 fg mL−1. The specificity test showed that the AFB1 immunosensor had no obvious cross-reaction with OTA, DON, ZEN, and FB1. The signal of this sensor decreased by 10.18% in 4 weeks indicating satisfactory stability, and its intra- and inter-laboratory reproducibility was 3.42~10.35% and 4.03%~12.11%, respectively. This biosensing system will open up new opportunities for the detection of AFB1 in food safety and environmental analysis and extend a wide range of applications in the analysis of other small molecules.

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References

  1. Adebo OA, Njobeh PB, Sidu S, Adebiyi JA, Mavumengwana V (2017) Aflatoxin B1 degradation by culture and lysate of a pontibacter specie. Food Control 80:99–103

    Article  CAS  Google Scholar 

  2. Ventura M, Gómez A, Anaya I, Díaz J, Broto F, Agut M, Comellas L (2004) Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromatography-tandem mass spectrometry. J Chromatogr A 1048(1):25–29

    CAS  PubMed  Google Scholar 

  3. 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):8873–8880

    Article  CAS  PubMed  Google Scholar 

  4. Yagati AK, Chavan SG, Baek C, Lee M, Min J (2018) Label-free impedance sensing of aflatoxin B1 with polyaniline nanofibers/Au nanoparticle electrode array. Sensors-Basel 18(5):1320

    Article  PubMed Central  CAS  Google Scholar 

  5. Goud KY, Reddy KK, Satyanarayana M, Kummari S, Gobi KV (2020) A review on recent developments in optical and electrochemical aptamer-based assays for mycotoxins using advanced nanomaterials. Microchim Acta 187(1):29

    Article  CAS  Google Scholar 

  6. Xue Z, Zhang Y, Yu W, Zhang J, Wang J, Wan F, Kim Y, Liu Y, Kou X (2019) Recent advances in aflatoxin B1 detection based on nanotechnology and nanomaterials-a review. Anal Chim Acta 1069(3):1–27

    Article  CAS  PubMed  Google Scholar 

  7. Ren X, Yan J, Wu D, Wei Q, Wan Y (2017) Nanobody-based apolipoprotein E immunosensor for point-of-care testing. Acs Sens 2(9):1267–1271

    Article  CAS  PubMed  Google Scholar 

  8. Tan Y, Chu X, Shen G, Yu R (2009) A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice. Anal Biochem 387(1):82–86

    Article  CAS  PubMed  Google Scholar 

  9. Zhang W, Dai Z, Liu X, Yang J (2018) High-performance electrochemical sensing of circulating tumor DNA in peripheral blood based on poly-xanthurenic acid functionalized MoS2 nanosheets. Biosens Bioelectron 105:116–120

    Article  CAS  PubMed  Google Scholar 

  10. Wang L, Sun Q, Liu Y, Lu Z (2016) Voltammetric determination of 4-chlorophenol using multiwall carbon nanotube/gold nanoparticle nanocomposite modified glassy carbon electrodes. RSC Adv 6(41):34692–34698

    Article  CAS  Google Scholar 

  11. Chen J, Bi H, Sun S, Tang Y, Zhao W, Lin T, Wan D, Huang F, Zhou X, Xie X, Jiang M (2013) Highly conductive and flexible paper of 1D silver-nanowire-doped graphene. ACS Appl Mater Interfaces 5(4):1408–1413

    Article  CAS  PubMed  Google Scholar 

  12. Wang J, Zhuo Y, Zhou Y, Yuan R, Chai Y (2015) Electrochemiluminescence immunosensor based on multifunctional luminol-capped AuNPs@Fe3O4 nanocomposite for the detection of mucin-1. Biosens Bioelectron 71:407–413

    Article  CAS  PubMed  Google Scholar 

  13. Yang H, Xu W, Liang X, Yang Y, Zhou Y (2020) Carbon nanotubes in electrochemical, colorimetric, and fluorimetric immunosensors and immunoassays: a review. Microchim Acta 187(4):206

    Article  CAS  Google Scholar 

  14. Fang X, Hua C, Wu C, Wang X, Shen L, Kong Q, Wang J, Hu Y, Wang Z, Chen L (2013) Synthesis and electrochemical performance of graphene-like WS2. Chem Eur J 19(18):5694–5700

    Article  CAS  PubMed  Google Scholar 

  15. Li J, Zhao Q, Tang Y (2016) Label-free fluorescence assay of S1 nuclease and hydroxyl radicals based on water-soluble conjugated polymers and WS2 nanosheets. Sensors-Basel 16(6):865

    Article  PubMed Central  CAS  Google Scholar 

  16. Zheliuk O, Lu J, Yang J, Ye J (2017) Monolayer superconductivity in WS2. Phys Status Solidi-R 11(9):1700245

    Article  CAS  Google Scholar 

  17. Cong C, Shang J, Wang Y, Yu T (2018) Optical properties of 2D semiconductor WS2. Adv Opt Mater 6(1):1700767

    Article  CAS  Google Scholar 

  18. Jing P, Yi H, Xue S, Chai Y, Yuan R, Xu W (2015) A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene-molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification. Anal Chim Acta 853(1):234–241

    Article  CAS  PubMed  Google Scholar 

  19. Su S, Sun H, Cao W, Chao J, Peng H, Zuo X, Yuwen L, Fan C, Wang L (2016) Dual-target electrochemical biosensing based on DNA structural switching on gold nanoparticle-decorated MoS2 nanosheets. ACS Appl Mater Interfaces 8(11):6826–6833

    Article  CAS  PubMed  Google Scholar 

  20. Muyldermans S, Atarhouch T, Saldanha J, Barbosa JARG, Hamers R (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng Des Sel 7:1129–1135

    Article  CAS  Google Scholar 

  21. Liu X, Wang D, Chu J, Xu Y, Wang W (2018) Sandwich pair nanobodies, a potential tool for electrochemical immunosensing serum prostate-specific antigen with preferable specificity. J Pharmaceut Biomed 158:361–369

    Article  CAS  Google Scholar 

  22. Rossotti MA, Pirez M, Gonzalez-Techera A, Cui Y, Bever CS, Lee KSS, Morisseau C, Leizagoyen C, Gee S, Hammock BD, González-Sapienza G (2015) Method for sorting and pairwise selection of nanobodies for the development of highly sensitive sandwich immunoassays. Anal Chem 87(23):11907–11914

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gong X, Zhu M, Li G, Lu X, Wan Y (2016) Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library. Anal Biochem 500(1):66–72

    Article  CAS  PubMed  Google Scholar 

  24. Saerens D, Frederix F, Reekmans G, Conrath K, Jans K, Brys L, Huang L, Bosmans E, Maes G, Borghs G, Muyldermans S (2005) Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing. Anal Chem 77(23):7547–7555

    Article  CAS  PubMed  Google Scholar 

  25. Dirks RM, Pierce NA (2004) Triggered amplification by hybridization chain reaction. P Natl Acad Sci U S A 101(43):15275–15278

    Article  CAS  Google Scholar 

  26. Lin R, Feng Q, Li P, Zhou P, Wang R, Liu Z, Wang Z, Qi X, Tang N, Shao F, Luo M (2018) A hybridization-chain-reaction-based method for amplifying immunosignals. Nat Methods 15(4):275–278

    Article  PubMed  CAS  Google Scholar 

  27. Xu W, Tian J, Shao X, Zhu L, Huang K, Luo Y (2016) A rapid and visual aptasensor for lipopolysaccharide detection based on the bulb-like triplex turn-on switch coupled with HCR-HRP nanostructures. Biosens Bioelectron 89(Pt 2):795–801

    PubMed  Google Scholar 

  28. Zhou X, Xue S, Jing P, Xu W (2016) A sensitive impedimetric platform biosensing protein: insoluble precipitates based on the biocatalysis of manganese (III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrinin in HCR-assisted dsDNA. Biosens Bioelectron 86:656–663

    Article  CAS  PubMed  Google Scholar 

  29. Li D, Cui Y, Morisseau C, Gee SJ, Bever CS, Liu X, Wu J, Hammock BD, Ying Y (2017) Nanobody based immunoassay for human soluble epoxide hydrolase detection using polymeric horseradish peroxidase (polyHRP) for signal enhancement: the rediscovery of polyHRP? Anal Chem 89(11):6248–6256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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(18):5221–5229

    Article  CAS  PubMed  Google Scholar 

  31. Zhang F, Liu B, Sheng W, Zhang Y, Liu Q, Li S, Wang S (2018) Fluoroimmunoassays for the detection of zearalenone in maize using CdTe/CdS/ZnS quantum dots. Food Chem 255:421–428

    Article  CAS  PubMed  Google Scholar 

  32. Zhang X, Song M, Yu X, Wang Z, Ke Y, Jiang H, Li J, Shen J, Wen K (2017) Development of a new broad-specific monoclonal antibody with uniform affinity for aflatoxins and magnetic beads-based enzymatic immunoassay. Food Control 79:309–316

    Article  CAS  Google Scholar 

  33. Heydari-Bafrooei E, Shamszadeh NS (2017) Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen. Biosens Bioelectron 91:284–292

    Article  CAS  PubMed  Google Scholar 

  34. Zhang K, Lu L, Wen Y, Xu J, Duan X, Zhang L, Hu D, Nie T (2013) Facile synthesis of the necklace-like graphene oxide-multi-walled carbon nanotube nanohybrid and its application in electrochemical sensing of azithromycin. Anal Chim Acta 787(13):50–56

    Article  CAS  PubMed  Google Scholar 

  35. Zhao W, Song C, Pehrsson PE (2002) Water-soluble and optically pH-sensitive single-walled carbon nanotubes from surface modification. J Am Chem Soc 124(42):12418–12419

    Article  CAS  PubMed  Google Scholar 

  36. Zeng Z, Yin Z, Huang X, Li H, He Q, Lu G, Boey F, Zhang H (2011) Single-layer semiconducting nanosheets: high-yield preparation and device fabrication. Angew Chem Int Ed 50:11093–11097

    Article  CAS  Google Scholar 

  37. Zheng J, Hu Y, Bai J, Ma C, Li J, Li Y, Shi M, Tan W, Yang R (2014) Universal surface-enhanced raman scattering amplification detector for ultrasensitive detection of multiple target analytes. Anal Chem 86(4):2205–2212

    Article  CAS  PubMed  Google Scholar 

  38. Zhu Q, Chai Y, Zhuo Y, Yuan R (2015) Ultrasensitive simultaneous detection of four biomarkers based on hybridization chain reaction and biotin-streptavidin signal amplification strategy. Biosens Bioelectron 68:42–48

    Article  CAS  PubMed  Google Scholar 

  39. Bhardwaj H, Pandey MK, Rajesh SG (2019) Electrochemical aflatoxin B1 immunosensor based on the use of graphene quantum dots and gold nanoparticles. Microchim Acta 186(8):592

    Article  CAS  Google Scholar 

  40. Lin Y, Zhou Q, Tang D (2017) Dopamine-loaded liposomes for in-situ amplified photoelectrochemical immunoassay of AFB1 to enhance photocurrent of Mn(2+)-doped Zn3(OH)2V2O7 nanobelts. Anal Chem 89(21):11803–11810

    Article  CAS  PubMed  Google Scholar 

  41. Lin Y, Zhou Q, Zeng Y, Tang D (2018) Liposome-coated mesoporous silica nanoparticles loaded with L-cysteine for photoelectrochemical immunoassay of aflatoxin B1. Microchim Acta 185(6):311

    Article  CAS  Google Scholar 

  42. Sun C, Liao X, Jia B, Shi L, Zhang D, Wang R, Zhou L, Kong W (2020) Development of a ZnCdS@ZnS quantum dots-based label-free electrochemiluminescence immunosensor for sensitive determination of aflatoxin B1 in lotus seed. Microchim Acta 187(4):236

    Article  CAS  Google Scholar 

  43. Wu L, Ding F, Yin W, Ma J, Wang B, Nie A, Han H (2017) From electrochemistry to electroluminescence: development and application in a ratiometric aptasensor for aflatoxin B1. Anal Chem 89(14):7578–7585

    Article  CAS  PubMed  Google Scholar 

  44. Shen Y, Pan D, Li G, Hu H, Xue H, Zhang M, Zhu M, Gong X, Zhang Y, Wan Y (2018) Direct immunoassay for facile and sensitive detection of small molecule aflatoxin B1 based on nanobody. Chem Eur J 24:9869–9876

    Article  PubMed  CAS  Google Scholar 

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Funding

This work was supported by Jiangxi Province Science Foundation for Youths (20171BAB214038), the Outstanding Young Talent Program of Jiangxi Province (20171BCB23042), the Graduate Innovative Special Fund Projects of Jiangxi Province (YC2019-S185), Jiangxi Provincial Academic and Technical Leaders Program (20182BCB22003), and National Natural Science Foundation of China (51662014, 51962007).

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Authors and Affiliations

  1. Key Lab for Agro-product Processing and Quality Control of Nanchang City, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045, China

    Xin Liu, Wenjun Wang, Zitong Zhao, Yi Han, Kaijie Tang & Dan Wang

  2. Department of Biomedical Engineering, Biosensor National Special Laboratory, Key Laboratory of Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, 310027, China

    Xin Liu

  3. Institute of Functional Materials and Agricultural Applied Chemistry, Jiangxi Agricultural University, Nanchang, 330045, China

    Yangping Wen

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  1. Xin Liu
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Correspondence to Wenjun Wang or Dan Wang.

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Liu, X., Wen, Y., Wang, W. et al. Nanobody-based electrochemical competitive immunosensor for the detection of AFB1 through AFB1-HCR as signal amplifier. Microchim Acta 187, 352 (2020). https://doi.org/10.1007/s00604-020-04343-2

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