Application of nanotechnology to pathogen detection and inactivation

Listeria monocytogenes is a psychrotrophic, Gram-positive pathogen frequently involved in outbreaks of food-borne disease in which milk, cheese, meat, and vegetables represent the incriminated foodstuffs. L. monocytogenes biofilms formed on food contact surfaces and processing environments are the major sources of contamination to ready-to-eat food products. Nanotechnology as an emerging new technology has many biological applications. The objectives of this study were to apply immunonanoparticles, carboxyl modified nanoparticles covalently bound with anti-L. monocytogenes, for L. monocytogenes detection and inactivation.; Immunonanopaticles and fluorescent immunonanoparticles were applied to indirect fluorescent immunoassay and direct fluorescent immunoassay, respectively, for sensitive detection of L. monocytogenes in biofilms. Immunonanoparticle-based immunoassay generated fluorescent intensity 16 times higher than antibody-based traditional immunoassay. In two-species biofilms, L. monocytogenes cells at different depths (0-3 microm) of biofilms were successfully detected by both immunonanoparticle-based indirect immunoassay and fluorescent immunonanoparticle-based direct immunoassay.; Immuno-magnetic-nanoparticle (IMNP)-based immunomagnetic separation (IMS) in combination with real-time PCR (RT-PCR) was used for a rapid and quantitative detection of L. monocytogenes in artificially contaminated milk. Capture efficiencies (CEs) by plating for IMNP-based IMS were 1.4~26 times higher than those of DynabeadsRTM-based IMS. When combined with RT-PCR, IMNP-based method, with a detection limit of 226 CFU/0.5 ml milk, was ca. 3 log magnitude more sensitive than DynabeadsRTM-based method. Cell numbers derived from IMNP-based RT-PCR were more than 25 times closer to the actual cell numbers present in milk samples than those from DynabeadsRTM based RT-PCR.; The antimicrobial activity of lysozyme-carrying immunonanoparticles against L. monocytogenes was studied. Enhanced antibacterial activity of lysozyme-carrying immunonanoparticles was achieved when anti-L. monocytogenes at the concentration of 0.04 microg/ml was used for coating nanoparticles and the resulting immunonanoparticles were coated with lysozyme for 6 h. Lysozyme-carrying immunonanoparticles (37 microg lysozyme/ml) exerted significantly (P < 0.05) higher anti-L. monocytogenes activities than lysozyme-carrying nanoparticles (37 microg lysozyme/ml), lysozyme solutions at higher concentrations (50 and 500 microg/ml). In conclusion, our results demonstrated that nanotechnology could significantly improve the current pathogen detection methods and enhance the antimicrobial activities of existing inactivation methods.