Carbon dot-enhanced nanobody-based fluorescence immunoassay for ultrasensitive Listeria monocytogenes detection in dairy products.

Listeria monocytogenes (L. monocytogenes), a psychrophilic Gram-positive bacterium, causes severe foodborne listeriosis outbreaks. However, rapid, accurate diagnostic assays for this pathogen in complex food matrices remain limited. Herein, we established a phage-displayed nanobody (Nb) library by immunizing a bactrian camel with inactivated L. monocytogenes. After biopanning, four specific Nbs were obtained with good specificity and thermal stability. Using soluble Nbs as capture antibodies and phage-displayed nanobodies as detecting antibodies, a fluorescent ELISA for L. monocytogenes was constructed by employing carbon dots as fluorescent probes based on the inner filter effect-mediated quenching. The developed assay exhibited a limit of detection of 3.21 × 103 CFU/mL, demonstrating a 144-fold enhancement over conventional ELISA, enabling highly sensitive and specific pathogen detection. In real sample analysis, the average recoveries ranged from 97.6 to 119.6 % and the relative standard deviation below 6.3 %, showcasing significant potential in food analysis applications.