An eco-friendly and ultrasensitive micro-orifice resistance assay for the detection of Listeria monocytogenes based on calcium carbonate signal probes harvested from discarded eggshells

The development of eco-friendly analytical methods for the detection of foodborne pathogens is of great importance for protecting human health and alleviating environmental pressure. Here, we demonstrate that a material (CaCO3 microparticles) prepared from discarded eggshells can be used as signal probes for micro-orifice resistance assay (MORA) and can be combined with ligation-mediated branching hybridization chain reaction (LB-HCR)-based signal-amplification technique (e-LMORA) for high sensitivity detection of Listeria monocytogenes. This strategy used LB-HCR-induced aggregation of streptavidin-modified CaCO3 microparticles, which were recognized by the particle counter for quantitative analysis of the target DNA. Compared with traditional MORA using polystyrene microspheres as signal probes, CaCO3 microparticles prepared from waste eggshells are more environmentally friendly. The combination of DNA-recognition probe, signal probes, and enzyme-free signal-amplification strategy not only reduces the cost synergistically, but also enables the biosensor to attain high sensitivity (limit of detection = 21 CFU/mL) due to its efficient signal amplification.