Polymer-based microfluidic chip for rapid and efficient immunomagnetic capture and release of Listeria monocytogenes.

Infections caused by foodborne pathogens such as Listeria monocytogenes pose a threat to public health while timely detection is challenging due to pathogen low numbers. The development of robust and efficient sample preparation techniques is crucial to improve detection sensitivity and workflow. Immunomagnetic separation using magnetic nanoparticles (MNPs) is attractive, as it can efficiently capture target cells. For food safety applications, a platform is needed to rapidly process large sample volumes, allowing capture and release of target bacteria conjugated to immunomagnetic nanoparticles (IMNPs). Herein, we demonstrate a method for magnetic capture and release of bacteria-IMNPs complex based on a 3D magnetic trap integrated on a polymeric microfluidic device. The 3D magnetic capture region consist of a dense array of high-aspect ratio (3 : 1) cylindrical pillars embossed in thermoplastic polymer and coated with soft ferromagnetic nickel by an electroless deposition technique. This allows the generation of strong and switchable magnetic capture regions due to the very low remanence of the nickel shell. We propose and validate an optimized configuration of capture regions for efficient localized capture and rapid release of MNPs and IMNPs conjugated to L. monocytogenes. A maximum recovery rate for MNPs corresponded to 91% while a maximum capture efficiency of 30% was obtained for live bacteria, with a minimum detectable sample concentration of ~10 cfu ml(-1) in 1 ml volume using plate-culture method. We believe that the flexible design and low-cost fabrication process of the proposed system will allow rapid sample preparation for applications beyond food and water safety, including point-of-care diagnosis.

A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis.

AIMS: A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated. METHODS AND RESULTS: Cryptic plasmids were mixed with an erythromycin resistance (Eryr) marker plasmid and introduced into a recipient strain by electroporation, followed by plasmid extraction of erythromycin-resistant transformants. By optimizing the ratio between the marker plasmid and the cryptic plasmids, an average of 20% cotransformants was obtained, including combinations of more than one cryptic plasmid. The marker plasmid pSA3 was easily eliminated from the cotransformed cells by subculture without selective pressure. CONCLUSION: This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids. SIGNIFICANCE AND IMPACT OF THE STUDY: The method facilitates the isolation of cryptic plasmids, helps in assigning functions to unknown plasmids and allows construction of food-grade lactococcal strains with new combinations of wild-type plasmids.