Enhanced nanobody-driven bioluminescent immunoassay for rapid parathion detection using engineered split-nanoluciferase

In this work, with parathion, a typical forbidden organophosphate pesticide as target drug, an enhanced nanobody-driven bioluminescent immunoassay based on the engineered split-nanoluciferase (NanoLuc) was proposed. Concretely, through labeling 11S and β10, two split-NanoLuc units onto the anti-parathion nanobody (Nb) VHH9 and the artificial antigen H1 coupled with carrier protein ovalbumin (H1-OVA) respectively, an NanoLuc Binary Technology (NanoBiT) system was firstly developed in the form of homogeneous immunoassay, in which the luminescence signal was produced by the reassembled NanoLuc after the combination of the 11S-fused VHH9 and β10-labeled H1-OVA. Subsequently, in order to enhance the signal-to-noise (S/N) ratio, a novel strategy of splitting 11S into two smaller subunits Δ11S and β9 was adopted so then an NanoLuc Ternary Technology (NanoTeT) system based on tri-part components of β9-fused VHH9, β10-labeled H1-OVA and Δ11S was successfully established. The results showed that the maximum half inhibition concentration (IC₅₀) for parathion can be as low as 2.04 ng/mL, 3.2-fold and 4.2-fold improved than that of the NanoBiT system and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Meanwhile, the detection range was from 0.19 ng/mL to 22.11 ng/mL. More importantly, this method required simply a one-step incubation with all reagents mixed together, and the total time used in detection was only 10 min, 7-fold faster than ic-ELISA. Finally, the average recoveries for vegetable samples were from 84.8% to 122% with the coefficient of variance (CV) below 15%. Overall, this study provides a new platform for homogeneous immunoassay of the small-molecule contaminants.