Lab-on-a chip platform captures bacteria, monitors growth and then detect. Dielectrophoresis (DEP) as part of the chip set up, helps concentrate bacterial cells on the silicon dioxide chip surface, pre-coated with pathogen specific antibodies or bio-receptor molecules for specificity. Low conductive growth media flown through the chip promote bacterial growth and impedance analyzer monitors resulting changes in the conductivity of the media. On chip PCR assay is now being developed to detect captured bacteria. Detection of target pathogens at low levels is possible in less than 6 h. Our contribution to this multidisciplinary project is to generate specific antibodies or bio-receptor molecules, media development, assay validation and microbiological analysis. Biochip platform has been optimized for L. monocytogenes detection. Our current effort is on Salmonella and Shiga-toxigenic E. coli.
Fiber optic biosensor:
Fundamental property of optical fiber is its ability to deliver the incoming light signal, and to act as a transducer of outgoing light. In fiber optic immunosensor, antibodies or bioreceptors in sandwich configuration detect pathogens and toxins. Captured target analyte on fiber optic waveguide is detected by using a fluorophore labeled antibody/bio-receptor. As laser is shined on the top of the fiber, it excites the fluorophore, generating evanescent wave, which returns to the detector. Signal is proportional to the amount of analyte bound to the fiber surface. The detection limit for three major foodborne pathogens, Salmonella, E. coli O157:H7 and L. monocytogenes is about 103 cells/ml. We have also demonstrated that all three pathogens can be detected simultaneously if present in the same sample.
Surface Plasmon Resonance (SPR) biosensor:
We are using SPR to study protein-protein interaction i.e., between Hsp60 and Listeria Adhesion Protein (LAP) in L. monocytogenes and between intimin and translocated intimin receptor (TIR) in E. coli O157:H7.
Develop and optimize reagents including antibodies, receptors, ligands, and microbiological growth media for biosensor applications
· Antibody development and characterization
· Influence of environmental or food–related stress on bacterial capture and detection
· Cloning and characterization of bio-receptor as capture molecule for foodborne pathogens
· Bacterial growth media development for biosensor-based detection
· Sample processing and preparation strategies using affinity based separation methods such as paramagnetic beads and ion exchange resins in a pathogen enrichment device (PED)
Understanding the molecular and cellular mechanism of intracellular Listeria monocytogenes colonization and translocation through epithelial barrier during intestinal phase of infection
Adhesion of L. monocytogenes to intestinal cells is an important initial event in Listeria pathogenesis. Our research group has discovered a 104-kDa Listeria adhesion protein, designated LAP that plays an important role during the intestinal phase of L. monocytogenes infection. LAP is present in all Listeria spp. including two newly reported listeriae; L. marthii and L. rocourtiae (unpublished) but absent in L. grayi. It functions as a housekeeping enzyme, an alcohol acetaldehyde dehydrogenase (AAD; lmo 1634) consisting of 866 amino acids with an N-terminal acetaldehyde dehydrogenase (ALDH), and a C-terminal alcohol dehydrogenase (ADH). A putative NAD+ binding domain is located between Gly427–Gly432, and an Fe2+ binding domain is located between Gly724–Gly742. Although it is present in both pathogenic and nonpathogenic Listeria species, only pathogenic bacteria secrete and re-associate LAP onto the cell surface to promote host cell interaction. The host cell receptor is a mitochondrial chaperonin called heat shock protein 60 (Hsp60). The N-terminal Gly224-Gly411 in ALDH domain interact with Hsp60. We showed that LAP–Hsp60 interaction promotes transepithelial translocation of L. monocytogenes through a paracellular route, suggesting an alternate strategy for bacteria to cross epithelial barriers during the intestinal phase of infection. Furthermore, L. monocytogenes infection at low dosage also increases Hsp60 expression, promoting enhanced LAP-mediated bacterial translocation.
Prevention and control strategies using probiotic bacteria and antimicrobial peptide loaded biocompatible nano carrier
· Application of recombinant probiotics to prevent adhesion and colonization of enteric pathogens
· Application of anti-microbial peptide loaded nano-carrier to control pathogens in food or intracellular compartments