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. 2023 May 25;13(6):577.
doi: 10.3390/bios13060577.

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification

Pengyan Guo  1 , Kaiyin Huang  1   2 , Zijian Chen  1 , Zhenlin Xu  1 , Aifen Ou  3 , Qingchun Yin  4 , Hong Wang  1 , Xing Shen  1 , Kai Zhou  1   2
Affiliations

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification

Pengyan Guo et al. Biosensors (Basel). .

Abstract

The advantages of genetic modification and preferable physicochemical qualities make nanobody (Nb) easy to develop a sensitive and stable immunosensor platform. Herein, an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) based on biotinylated Nb was established for the quantification of diazinon (DAZ). The anti-DAZ Nb, named Nb-EQ1, with good sensitivity and specificity, was obtained from an immunized library via a phage display technique, where the molecular docking results indicated that the hydrogen bond and hydrophobic interactions between DAZ and complementarity-determining region 3 and framework region 2 in Nb-EQ1 played a critical role in the Nb-DAZ affinity processes. Subsequently, the Nb-EQ1 was further biotinylated to generate a bi-functional Nb-biotin, and then an ic-CLEIA was developed for DAZ determination via signal amplification of the biotin-streptavidin platform. The results showed that the proposed method based on Nb-biotin had a high specificity and sensitivity to DAZ, with a relative broader linear range of 0.12-25.96 ng/mL. After being 2-folds dilution of the vegetable samples matrix, the average recoveries were 85.7-113.9% with a coefficient of variation of 4.2-19.2%. Moreover, the results for the analysis of real samples by the developed ic-CLEIA correlated well with that obtained by reference method GC-MS (R2 ≥ 0.97). In summary, the ic-CLEIA based on biotinylated Nb-EQ1 and streptavidin recognition demonstrated itself to be a convenient tool for the quantification of DAZ in vegetables.

Keywords: diazinon; indirect competitive chemiluminescence enzyme immunoassay; nanobody; phage display; signal amplification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The affinity of two positive phage clones of Nb-EQ1 and Nb-EQ2 and their amino acid sequence alignment.
Figure 2
Figure 2
The comparation of stabilities of Nb-EQ1 and DAZ-mAb at (a,b) different pHs, (c) temperatures, and (d–f) in the presence of an organic solvent.
Figure 3
Figure 3
Structure simulation and docking analysis of Nb-EQ1 and DAZ. (a) The root-mean-square error; (b,c) three-dimensional diagram of docking plot; (d) two-dimensional diagram of interaction plot.
Scheme 1
Scheme 1
Schematic diagram of ic-CLEIA platform based on Nb-biotin and streptavidin recognition.
Figure 4
Figure 4
Effect of physicochemical parameters on ic-CLEIA performances. (a) Coating antigen/antibody concentration, (b) PBS concentration, (c) pH value, (d) tween-20 concentration.
Figure 5
Figure 5
The (a) standard curve of DAZ assessed by ic-CLEIA and (b) cross-reactivities of the obtained Nb with DAZ and its analogues.
Figure 6
Figure 6
Matrix effect of vegetable samples on the ic-CLEIA performance (n = 3); (a) cucumber, (b) lettuce, (c) cabbage.
See this image and copyright information in PMC

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