Recombinant Peptidomimetic-Nano Luciferase Tracers for Sensitive Single-Step Immunodetection of Small Molecules

Phage borne peptides isolated from phage libraries have proven to be valuable reagents for the development of small-molecule immunoassays. However, the large size, low diffusion rate, and biological nature of the phage particles create some limitations to their use and require secondary reagents for its detection. In this work, we explore the use of the Nano luciferase (NanoLuc) as a fusion partner to generate recombinant tracers for immunoassay development. The imidaclothiz peptidomimetic C2-15 that specifically binds to the anti-imidaclothiz monoclonal antibody (mAb) 1E₇ was fused to NanoLuc, both at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15). NanoLuc-C2-15 showed better performance than C2-15-NanoLuc and was adopted to develop a bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA) for imidaclothiz. The luminescence signal of NanoLuc-C2-15 rapidly reaches high intensity with slow attenuation, which enabled one to capture the BLLFIA readout by using a smartphone without an external light source. The IC₅₀ of the BLEIA and BLLFIA were 3.3 ± 0.2 and 6.4 ± 0.4 ng mL^-1, respectively. Both immunoassays exhibited good accuracy for the detection of imidaclothiz in environmental and agricultural samples.