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. 2021 Jan 19;19(1):27.
doi: 10.1186/s12951-021-00773-z.

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1

Sha Wu  1   2 , Fengfei Ma  1   2 , Jinxin He  1   2 , Qing X Li  3 , Bruce D Hammock  4 , Jiesheng Tian  5 , Ting Xu  6   7
Affiliations

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1

Sha Wu et al. J Nanobiotechnology. .

Abstract

Background: Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil.

Results: Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%.

Conclusions: The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

Keywords: Fipronil; Immunoassay; Magnetosome; Magnetospirillum gryphiswaldense; Nanobody.

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Conflict of interest statement

The authors have declared that no competing interests exist. All data and materials are available and agreed to publish.

Figures

Fig. 1
Fig. 1
Construction of recombinant strains biomineralizing Nb-magnetosomes. a The schematic diagram of recombinant strain construction. b Colony PCR of CF. genes in group 1 were amplified by using Fip-F and Fip-R (left to right: marker, negative control, Nb F1, and CF); genes in group 2 were amplified by P1 and P3 (left to right: marker, negative control, WT MSR-1, and CF); genes in group 3 were amplified by P2 and P4 (left to right: marker, negative control, WT MSR-1, and CF). c Colony PCR of CF+ . The first lane and the last lane were negative control and marker, respectively; the rest of lanes were sampling. Primers used were pBBR-F and pBBR-R. d Colony PCR of CFFF. The first and the second lane were marker and negative control, respectively; the rest of lanes were sampling. Primers used were mamF and Fip-R. All primers were listed in Additional file 1: Table S2
Fig. 2
Fig. 2
The growth (OD565) and magnetic response (Cmag values) curves of CF (a), CF + (b) and CFFF (c) fed-batch cultured in a fermenter
Fig. 3
Fig. 3
Non-competitive ELISAs based on three Nb-magnetosomes (a) and competitive ELISAs for fipronil based on Nb-magnetosomes from CF (b); CF+ (c) and CFFF (d). “*”: significant difference between WT and CF (p < 0.05); “***”: large significant difference between WT and CFFF (p < 0.01)
Fig. 4
Fig. 4
Competitive ELISAs for fipronil using Nb-magnetosomes treated with H2O2 (a) or a mixture of GSH and GSSG (b). “*”: significant difference (p < 0.05)
Fig. 5
Fig. 5
Calibration curve of Nb-magnetosome-based ELISA for fipronil. The data are average of three replicates
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