Multivalent bifunctional nanobody to enhance the sensitivity of direct competitive chemiluminescence immunoassay for the detection of microcystin LR in lake water.

Microcystin-LR (MC-LR), a toxic cyanobacterial toxin in freshwater, poses significant health and ecological risks due to its ability to induce cell apoptosis and liver damage. Sensitive detection of MC-LR is crucial for public health and water safety. In this work, we engineered a multivalent bifunctional nanobody (A2.3-C4-SBP) by fusing the anti-MC-LR nanobody gene (A2.3) with self-assembling peptides (C4) and a streptavidin-binding peptide (SBP). A2.3-C4-SBP was directionally immobilized on the ELISA microplate via streptavidin-mediated to develop a multivalent bifunctional nanobody-based chemiluminescent immunoassay (MBN-CLIA) for MC-LR detection in lake water. The IC of the A2.3-C4-SBP heptamer based CLIA was 5.80 ng/mL, and the LOD (IC) was 0.33 ng/mL, which were 9.51-fold and 1.82-fold lower, respectively, than those of the A2.3-SBP monomer based CLIA. Additionally, the IC and LOD were 1.26-fold and 1.82-fold lower, respectively, than those of the A2.3-C4-SBP heptamer without streptavidin-mediated directional immobilization. In summary, this work developed a sensitive, rapid and simple immunoassay for the detection of MC-LR in lake water based on multivalent bifunctional nanobodies. Furthermore, the proposed combined strategy of nanobody multimerization and directed immobilization is simple to operate and has great potential to improve the sensitivity and signal amplification of various immunoassays.