Engineering a nanobody-alkaline phosphatase bifunctional probe for enhanced immunoassay performance toward microcystin-LR.

Eutrophication caused by excessive nutrient discharge has led to the frequent occurrence of cyanobacterial blooms, during which toxic metabolites such as microcystins (MCs) are released into aquatic environments. Among these, microcystin-LR (MC-LR) is the most widespread and toxic congener, posing serious threats to both ecological safety and human health. In this study, a novel bifunctional detection probe (A2.3-AP) was developed by genetically fusing a MC-LR-specific nanobody (A2.3) with alkaline phosphatase (AP) via a flexible (GGGGS)3 linker. The A2.3-AP bifunctional detection probe integrates antigen recognition and enzymatic catalysis within a single molecule, thereby simplifying the immunoassay procedure and improving detection performance. Based on this bifunctional probe, a one-step indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established for the rapid and sensitive determination of MC-LR. Compared with conventional ELISA, the proposed method reduced the assay time by approximately one hour and improved the detection sensitivity threefold, achieving a detection limit of 12.47 ng mL-1. Lake water samples were used to verify the matrix tolerance of the assay, and spiked samples were cross-validated by HPLC to confirm its accuracy. The A2.3-AP based immunoassay provides a high-performance analytical tool for the prevention and control of MC-LR contamination events and offers a new approach for improving traditional enzyme-linked immunoassay methods.