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

Abstract

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.

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Acknowledgments

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.

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Correspondence to Yanfei Shen or Yakun Wan.

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Min Zhu and Min Li contributed equally to this work.

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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). https://doi.org/10.1007/s00604-015-1602-9

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Keywords

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