Plasmonic nanoparticle-based colorimetric sensor for detecting ribonucleases as biomarkers of Listeria spp

Selective cleavage of oligonucleotide probes by bacterial nucleases has emerged as a promising strategy for detecting foodborne pathogens. In this study, we applied this approach to enable rapid, colorimetric detection of Listeria monocytogenes in fresh leafy vegetables and raw sheep milk, two common sources of foodborne human listeriosis. We optimized (i) culture conditions to maximize nuclease production; (ii) probe design, including oligonucleotide sequence and chemical modifications, for selective gold nanoparticle aggregation; and (iii) experimental parameters of pH, temperature, and ionic strength for efficient reaction kinetics. Proteomic and size-exclusion chromatography analyses identified L. monocytogenes Ribonuclease II (29.349 kDa, encoded by rnhB) as the nuclease responsible for probe cleavage. Results from L. monocytogenes-spiked samples of ready-to-eat lettuce (fourth-range) and high-fat, high-Mg⁺⁺ sheep milk indicate that this method represents a rapid, easy-to-perform, and cost-effective diagnostic tool for pathogen screening in food and environmental samples. Overall, these findings highlight the enormous potential of nucleases as bacterial biomarkers in the fields of food security and health.