Biosynthetic Toxin Mimics Meet Nanophotonics: A Rapid, Label-Free Biosensor Platform for Real-Time Detection of Bongkrekic Acid in Urine.

Bongkrekic acid (BA) is a highly toxic, heat-stable bacterial toxin associated with severe foodborne outbreaks, particularly in Southeast Asia, with reported mortality rates of up to 100%. Despite its clinical relevance, current detection methods rely on complex, laboratory-based analytical techniques, thereby limiting the ability to reach a rapid diagnosis. Here, we present a novel strategy for BA detection that employs a recombinant antigen mimic generated by fusing a phage-display-selected BA epitope to maltose-binding protein (MBP), enabling safe, reproducible, and scalable immunoassay development without handling the native toxin. The BA-MBP fusion protein was used as a coating antigen in a competitive immunoassay (ic-ELISA), demonstrating sensitive antibody recognition with an assay IC50 of 6.24 ng/mL. This approach was further translated into a nanophotonic label-free biosensing platform to demonstrate the potential for rapid diagnostics in human samples. A single-step competitive biosensor assay provided an IC50 of 0.64 ng/mL and a limit of detection of 0.12 ng/mL, representing a 10-fold improvement over the ic-ELISA using conventional conjugates and providing a performance comparable to LC-MS/MS. The method was successfully validated in spiked blind urine samples, highlighting its applicability in clinically relevant matrices. The combination of BA-MBP fusion conjugate with photonic biosensing establishes a robust, safe, and scalable approach for rapid, reliable BA detection and quantification, with strong potential for point-of-care diagnostics and decentralized toxin monitoring.