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

• Published in: Biosensors (Basel) 2023-05, Vol.13 (6), p.577
• Main Authors: Guo, Pengyan, Huang, Kaiyin, Chen, Zijian, Xu, Zhenlin, Ou, Aifen, Yin, Qingchun, Wang, Hong, Shen, Xing, Zhou, Kai
• Format: Article
• Language: English
• Subjects: Agricultural research; Amplification; Antibodies; Antigens; Biosensing Techniques - methods; Biotin; Biotin - chemistry; Chemiluminescence; Cloning; Coefficient of variation; Complementarity-determining region 3; Diazinon; Dilution; E coli; Enzyme immunoassay; Enzyme-Linked Immunosorbent Assay - methods; Enzymes; Fungicides; Genetic modification; Globular proteins; Hydrogen bonds; Hydrophobicity; Immunization; Immunoassay; Immunoassay - methods; Immunoenzyme technique; Immunoenzyme Techniques; Immunosensors; indirect competitive chemiluminescence enzyme immunoassay; Insecticides; Laboratories; Luminescence; Measurement; Methods; Molecular docking; Molecular Docking Simulation; Molecular weight; Nanobodies; nanobody; Phage display; Sensitivity; signal amplification; Streptavidin; Streptavidin - chemistry; Testing; Vegetables; Viral antibodies
• ISSN: 2079-6374; 2079-6374
• DOI: 10.3390/bios13060577

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 (R ≥ 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.