Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline


We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng∙mL-1 and a 0.07 ng∙mL-1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin.

Nanobodies specific to Cry1Ab toxin were isolated from an immunized camel. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay with horseradish peroxidase being used to produce polyaniline, which can be easily detected by differential pulse voltammetry.

This is a preview of subscription content, access via your institution.

Fig. 1
Scheme 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Soberon M, Gill SS, Bravo A (2009) Signaling versus punching hole: how do bacillus thuringiensis toxins kill insect midgut cells? Cell Mol Life Sci: CMLS 66(8):1337–1349. doi:10.1007/s00018-008-8330-9

    CAS  Article  Google Scholar 

  2. 2.

    Raybould A, Caron-Lormier G, Bohan DA (2011) Derivation and interpretation of hazard quotients to assess ecological risks from the cultivation of insect-resistant transgenic crops. J Agric Food Chem 59(11):5877–5885. doi:10.1021/jf1042079

    CAS  Article  Google Scholar 

  3. 3.

    Lemaux PG (2008) Genetically engineered plants and foods: a scientist's analysis of the issues (part I). Annu Rev Plant Biol 59:771–812. doi:10.1146/annurev.arplant.58.032806.103840

    CAS  Article  Google Scholar 

  4. 4.

    James C, International Service for the Acquisition of Agri-Biotech Applications. Global status of commercialized biotech/GM crops, 2009 : brief 41. ISAAA briefs, vol no 41

  5. 5.

    Gassmann AJ, Petzold-Maxwell JL, Keweshan RS, Dunbar MW (2011) Field-evolved resistance to Bt maize by western corn rootworm. PLoS One 6(7), e22629. doi:10.1371/journal.pone.0022629

    CAS  Article  Google Scholar 

  6. 6.

    Whiting SA, Strain KE, Campbell LA, Young BG, Lydy MJ (2014) A multi-year field study to evaluate the environmental fate and agronomic effects of insecticide mixtures. Sci Total Environ 497–498:534–542. doi:10.1016/j.scitotenv.2014.07.115

    Article  Google Scholar 

  7. 7.

    Zhang Y, Lai C, Su R, Zhang M, Xiong Y, Qing H, Deng Y (2012) Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18O stable isotope dilution. Analyst 137(11):2699–2705. doi:10.1039/c2an35383k

    CAS  Article  Google Scholar 

  8. 8.

    Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev: MMBR 62(3):775–806

    CAS  Google Scholar 

  9. 9.

    Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev: MMBR 62(3):807–813

    CAS  Google Scholar 

  10. 10.

    Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P (2011) Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol J 9(3):283–300. doi:10.1111/j.1467-7652.2011.00595.x

    CAS  Article  Google Scholar 

  11. 11.

    De Meyer T, Muyldermans S, Depicker A (2014) Nanobody-based products as research and diagnostic tools. Trends Biotechnol 32(5):263–270. doi:10.1016/j.tibtech.2014.03.001

    Article  Google Scholar 

  12. 12.

    De Genst E, Silence K, Decanniere K, Conrath K, Loris R, Kinne J, Muyldermans S, Wyns L (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 103(12):4586–4591. doi:10.1073/pnas.0505379103

    Article  Google Scholar 

  13. 13.

    Choi O, Lee Y, Han I, Kim H, Goo E, Kim J, Hwang I (2013) A simple and sensitive biosensor strain for detecting toxoflavin using beta-galactosidase activity. Biosens Bioelectrons 50:256–261. doi:10.1016/j.bios.2013.06.058

    CAS  Article  Google Scholar 

  14. 14.

    Lai G, Yan F, Wu J, Leng C, Ju H (2011) Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited by gold nanoparticles and enzymatic reaction. Anal Chem 83(7):2726–2732. doi:10.1021/ac103283p

    CAS  Article  Google Scholar 

  15. 15.

    Muguruma H, Hoshino T, Nowaki K (2014) Electronically type-sorted carbon nanotube-based electrochemical biosensors with glucose oxidase and dehydrogenase. ACS Appl Mater Interfaces. doi:10.1021/am506758u

    Google Scholar 

  16. 16.

    Tang N, Zheng J, Sheng Q, Zhang H, Liu R (2011) A novel H2O2 sensor based on the enzymatically induced deposition of polyaniline at a horseradish peroxide/aligned single-wall carbon nanotubes modified Au electrode. Analyst 136(4):781–786. doi:10.1039/c0an00379d

    CAS  Article  Google Scholar 

  17. 17.

    Lai G, Zhang H, Tamanna T, Yu A (2014) Ultrasensitive immunoassay based on electrochemical measurement of enzymatically produced polyaniline. Anal Chem 86(3):1789–1793. doi:10.1021/ac4037119

    CAS  Article  Google Scholar 

  18. 18.

    Sheng Q, Zheng J (2009) Bienzyme system for the biocatalyzed deposition of polyaniline templated by multiwalled carbon nanotubes: a biosensor design. Biosens Bioelectrons 24(6):1621–1628. doi:10.1016/j.bios.2008.08.029

    CAS  Article  Google Scholar 

  19. 19.

    Yu H, Yan F, Dai Z, Ju H (2004) A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chitosan-membrane-modified screen-printed carbon electrode. Anal Biochem 331(1):98–105. doi:10.1016/j.ab.2004.03.042

    CAS  Article  Google Scholar 

  20. 20.

    Li H, Yan J, Ou W, Liu H, Liu S, Wan Y (2015) Construction of a biotinylated cameloid-like antibody for lable-free detection of apolipoprotein B-100. Biosens Bioelectrons 64:111–118. doi:10.1016/j.bios.2014.08.060

    CAS  Article  Google Scholar 

  21. 21.

    Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y (2014) Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2. Nanoscale Res Lett 9(1):528. doi:10.1186/1556-276X-9-528

    Article  Google Scholar 

  22. 22.

    Wang P, Li G, Yan J, Hu Y, Zhang C, Liu X, Wan Y (2014) Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin. Toxicon: Off J Int Soc Toxinol 92:186–192. doi:10.1016/j.toxicon.2014.10.024

    CAS  Article  Google Scholar 

  23. 23.

    Vincke C, Gutierrez C, Wernery U, Devoogdt N, Hassanzadeh-Ghassabeh G, Muyldermans S (2012) Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol Biol 907:145–176. doi:10.1007/978-1-61779-974-7_8

    CAS  Article  Google Scholar 

  24. 24.

    Mu B, Huang X, Bu P, Zhuang J, Cheng Z, Feng J, Yang D, Dong C, Zhang J, Yan X (2010) Influenza virus detection with pentabody-activated nanoparticles. J Virol Methods 169(2):282–289. doi:10.1016/j.jviromet.2010.07.024

    CAS  Article  Google Scholar 

  25. 25.

    Zhang X, Liu Y, Zhang C, Wang Y, Xu C, Liu X (2012) Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin. Ecotoxicol Environ Saf 81:84–90. doi:10.1016/j.ecoenv.2012.04.021

    CAS  Article  Google Scholar 

  26. 26.

    Zhu M, Gong X, Hu Y, Ou W, Wan Y (2014) Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1. J Transl Med 12(1):352. doi:10.1186/s12967-014-0352-5

    Article  Google Scholar 

  27. 27.

    Peng Y, Yi G, Gao Z (2010) A highly sensitive microRNA biosensor based on ruthenium oxide nanoparticle-initiated polymerization of aniline. Chem Commun 46(48):9131–9133. doi:10.1039/c0cc01990a

    CAS  Article  Google Scholar 

  28. 28.

    Liu Y, Liu Y, Feng H, Wu Y, Joshi L, Zeng X, Li J (2012) Layer-by-layer assembly of chemical reduced graphene and carbon nanotubes for sensitive electrochemical immunoassay. Biosens Bioelectrons 35(1):63–68. doi:10.1016/j.bios.2012.02.007

    CAS  Article  Google Scholar 

  29. 29.

    Puttharugsa C, Wangkam T, Huangkamhang N, Gajanandana O, Himananto O, Sutapun B, Amarit R, Somboonkaew A, Srikhirin T (2011) Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody. Biosens Bioelectrons 26(5):2341–2346. doi:10.1016/j.bios.2010.10.007

    CAS  Article  Google Scholar 

  30. 30.

    Wang JM, Chen XP, Liang YY, Zhu HJ, Ding JT, Peng YF (2014) Influence of transgenic rice expressing a fused Cry1Ab/1Ac protein on frogs in paddy fields. Ecotoxicology 23(9):1619–1628. doi:10.1007/s10646-014-1301-z

    Article  Google Scholar 

  31. 31.

    Adel-Patient K, Guimaraes VD, Paris A, Drumare MF, Ah-Leung S, Lamourette P, Nevers MC, Canlet C, Molina J, Bernard H, Creminon C, Wal JM (2011) Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse. PLoS One 6(1), e16346. doi:10.1371/journal.pone.0016346

    Article  Google Scholar 

  32. 32.

    Ming H, Wang M, Yin H (2014) Detection of Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance immunosensor. Anal Biochem 468C:59–65. doi:10.1016/j.ab.2014.09.014

    Google Scholar 

  33. 33.

    Paul V, Steinke K, Meyer HH (2008) Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810). Anal Chim Acta 607(1):106–113. doi:10.1016/j.aca.2007.11.022

    CAS  Article  Google Scholar 

  34. 34.

    Sims SR, Holden LR (1996) Insect bioassay for determining soil degradation of Bacillus thuringiensis subsp kurstaki CryIA(b) protein in corn tissue. Environ Entomol 25(3):659–664

    Article  Google Scholar 

  35. 35.

    Chen C, Wu J (2012) A fast and sensitive quantitative lateral flow immunoassay for Cry1Ab based on a novel signal amplification conjugate. Sensors 12(9):11684–11696. doi:10.3390/s120911684

    CAS  Article  Google Scholar 

Download references


This work was supported by grants from Jiangsu Nanobody Engineering and Research Center of China (2015–06), Program for New Century Excellent Talents in University (NCET-20130127), National Natural Science Foundation of China (grant numbers 31271365, 31471216 and 21305065), the Key Program from the Natural Science Foundation of Jiangsu province (BK20130788) and the Fundamental Research Funds for the Central Universities. The work also supported by the Natural Science Foundation of China (U1301214), Guangdong Natural Science Foundation (S2013030013338).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Min Zhu, Min Li carried out the experiments and participated in the drafting of the manuscript. Guanghui Li participated in the library construction. Hong Liu performed the SPR assay to evaluate the affinity of nanobody. Zikai Zhou and Hongtao Lei participated in the detection analysis. Yanfei Shen and Yakun Wan participated in the design of the study. All authors read and approved the final manuscript.

Author information



Corresponding authors

Correspondence to Yanfei Shen or Yakun Wan.

Additional information

Min Zhu and Min Li contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.


(DOC 466 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhu, M., Li, M., Li, G. et al. Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline. Microchim Acta 182, 2451–2459 (2015).

Download citation


  • Nanobody
  • Polyaniline
  • Cry1Ab toxin
  • Sandwich immunoassay
  • Immunosensor
  • Differential pulse voltammetry