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. 2023 Sep 6;71(35):13137-13146.
doi: 10.1021/acs.jafc.3c03408. Epub 2023 Aug 23.

Development of a Biotinylated Nanobody-Based Gold Nanoparticle Immunochromatographic Assay for the Detection of Procymidone in Crops

Min-Ling Liu  1 , Xiao-Ting He  1 , Zhen-Lin Xu  1 , Hao Deng  2 , Yu-Dong Shen  1 , Lin Luo  1 , Xing Shen  1 , Zi-Jian Chen  3 , Bruce Hammock  4 , Hong Wang  1
Affiliations

Development of a Biotinylated Nanobody-Based Gold Nanoparticle Immunochromatographic Assay for the Detection of Procymidone in Crops

Min-Ling Liu et al. J Agric Food Chem. .

Abstract

A heavy-chain antibody (VHH) library against procymidone (PRM) was constructed via immunizing Bactrian camels. Through careful biopanning, seven nanobodies (Nbs) with different sequences were obtained. The variability in their performance was primarily attributed to the amino acid differences in complementarity-determining region 3 (CDR3), as analyzed by molecular docking. The Nb exhibiting the highest sensitivity, named NbFM5, was biotinylated and conjugated to streptavidin-labeled gold nanoparticles to preserve the epitope's activity and prevent a decrease in sensitivity due to traditional random electrostatic adsorption. Subsequently, a simple and sensitive immunochromatographic assay (ICA) was developed for rapid detection of PRM based on biotinylated Nb (btNb). The developed btNb-ICA showed a cut-off value of 200 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 6.04 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.88 ng/mL. The recoveries in actual samples of crops ranged from 82.2 to 117.3%, aligning well with the results obtained from GC-MS/MS (R2 = 0.995). In summary, the developed btNb-ICA demonstrated high specificity and good accuracy for the rapid detection of PRM residues in vegetables. The total analysis time from preparing the sample to obtaining the result was less than 25 min.

Keywords: biotinylation; immunochromatographic assay; nanobodies; procymidone; streptavidin.

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Conflict of interest statement

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Identification of serum subtypes isolated from serum in Bactrian camels and construction of Nb genes library. (a) Curves of antigen-binding capacity; (b) Curves of inhibition rate; (c) Isolation and identification of serum subtypes in Bactrian camels; (d) SDS-PAGE of serum subtypes. M: marker; 1: serum; 2: IgG1 elution; 3: IgG2 elution; 4: IgG3 elution; (e) Extraction of the total RNA; (f-g) The amplification of the VHH genes; (h) The second round of PCR and the digestion of pcomb3xSS.
Figure 2
Figure 2
Identification of Nbs with different sequences. (a) amino acid sequences of NbFM1 ~ NbFM7; (b) SDS-PAGE; (c) The amount of expression; (d) Identification of NbFM1 ~ NbFM7 by ic-ELISA.
Figure 3
Figure 3
Molecular docking between PRM and anti-PRM NbFM5/NbFM7. (a-b) The docking results between NbFM5 and PRM; (c) The interaction forces between NbFM5 and PRM; (d) The electrostatic potential energy generated by NbFM5-PRM; (e-f) The docking results between NbFM7 and PRM; (g) The interaction forces between NbFM7 and PRM; (h) The electrostatic potential energy generated by NbFM7-PRM.
Figure 4
Figure 4
Preparation of btNb and development of btNb-ICA; (a) Directed biotinylated gene design principle; (b) Directed btNb preparation and identification curve; M: Marker; 1: NbFM5; 2: btNb; (c) The principle of targeted labeling of anti-PRM btNb and detection of btNb-ICA; (d) The calibration curves of the btNb-ICA; (e) The stability test in 45°C of the btNb-ICA.
Figure 5
Figure 5
Optimization of extraction method and the sensitivity of btNb-ICA. (a) The effect of acetonitrile content in direct dilution pretreatment on detection sensitivity based on btNb-ICA; (b) The influence of different sample pretreatment methods on the recoveries of btNb-ICA; (c) The accuracy of btNb-ICA was verified by GC-MS/MS (n=3).
Scheme 1.
Scheme 1.
Preparation of anti-PRM Nbs and development of immunochromatography. (Number of animal ethical review certificate: 2020f064)
See this image and copyright information in PMC

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