Text Box: Research Text Box: Development of biosensor–based methods for pathogen detection 1. Antibody-based sensors Sample preparation: Success of biosensor tools requires efficient separation and extraction of bacterial cells from a complex food matrix. We have developed an immunobead separation method to capture and concentrate L. monocytogenes from a complex food system for detection of low numbers of bacteria. Also, a device is currently under development that will allow simultaneous sample enrichment and detection to improve bacterial cell recovery and direct detection with conventional or biosensor tools. Antibodies and performance: Antibodies are essential key molecules for most of our biosensor applications. In the past we have developed several monoclonal antibodies (MAb) against Listeria monocytogenes that are useful for most applications; however, we are continuing our efforts to develop more specific antibodies. Taking advantage of the genome sequence of L. monocytogenes, proteomic approaches were employed to develop specific antibodies for use with the biosensor applications. Several antibodies are now evaluated for their specificity, sensitivity and performance with biosensor tools. Similar strategies are used to develop antibodies against Salmonella enterica. Ability of the antibodies to react with stressed or injured target bacterial cells from food products or the enrichment media determines the success of any immunoassays or immunosensors. We are employing proteomic approaches to understand the mechanism for optimum expression of antibody-specific antigens in target cells under various stress conditions. Fiber optic biosensor: Using Listeria specific-antibodies, a fiber optic probe had been developed that could sensitively detect low levels (~103 cells) of L. monocytogenes in 2-3 hours. The ultimate goal is to develop a system to detect Salmonella, E. coli and L. monocytogenes concurrently. Surface Plasmon Resonance (SPR) biosensor: We are continuing our efforts in developing enhanced surface plasmon resonance (SPR) sensors using gold nanoparticles and modifications to existing technologies in an effort to increase the sensitivity of the current sensing platforms. Current projects include screening and characterization of aptamers for its suitability to detect food-borne pathogens. Projects are also under way for the development of a multi-pathogen detection assay using fiber optic and SPR biosensors. Our group is also using these optical biosensors to study protein-protein interaction eg., between Hsp-60 and LAP protein in L. monocytogenes Biochip: In collaboration with engineers from the School of Agriculture and the School of Engineering at Purdue University, a biochip sensor to detect viable L. monocytogenes from food is currently under development. The basic principle of this biosensor is to capture Listeria cells on the surface of the platinum and silicon dioxide chip using an antibody that is specific for L. monocytogenes and subsequent impedance measurement of bacterial cell growth. Substantial progress has been made towards the adsorption of antibodies and impedance-based bacterial growth measurement on-chip. Bacterial Rapid Detection using Optical Light Scattering (BARDOT) We have developed an optical light scattering instrument, in collaboration with school of mechanical engineering and used it for scanning bacterial colonies grown on an agar surface. The technology produces unique scatter patterns for different bacterial species. We have also developed suitable image analysis software in collaboration with the school of biomedical sciences which can identify the features in the scatter patterns and group them accordingly. We have observed 91-100% specificity in detecting bacteria based on its unique scatter images. 2. Cytopathogenicity assay for Listeria, Bacillus and E. coli O157:H7 Cell- based sensor for Listeria: Pathogenic potential of foodborne bacteria can be determined by assessing interaction of bacteria with an appropriate mammalian cell line. Bhunia and his co-workers discovered that certain hybrid B-lymphocytes are highly susceptible to pathogenic Listeria species. Using this cell line, an alkaline phosphatase -based colorimetric assay in a 96-well microtiter plate format has been developed that detects cytotoxicity in 1-2 h. To use cell-based sensor for onsite product testing, a portable system using hydrogel-collagen matrix is under development. Mechanism of B-cell cytotoxicity - Cytotoxic effect on hybrid B-lymphocytes was found to be due to the action of listeriolysin or phospholipase C, which cause apoptosis. The general belief is that B cells have no role in immunity against Listeria infection; however, these experiments provided clues that B-lymphocytes possibly are targets for L. monocytogenes infection in vivo. To examine this hypothesis, human B lymphoma and primary murine B cells were tested and the cells were killed by apoptosis, suggesting B cell populations probably are affected during Listeria infection in vivo.  Pratik and Dr. Bhunia: testing a cell-based sensor prototype Cytotoxicity assay for E. coli O157:H7: Kidney cells (Vero) derived from monkey are highly sensitive to pathogenic actions of toxins from enterohemorrhagic E. coli (EHEC) including O157:H7 strains. Conventional Vero cell assay requires at least 3-4 days. An enzyme (lactate dehydrogenase) - based assay was developed that can sensitively detect toxin producing EHEC from non-toxin producing strains in 12-16 hours. This system was thoroughly tested with E. coli from food and environmental sources. Cytotoxicity assay for Bacillus cereus: Bacillus species causing food-borne disease produce multiple toxins eliciting gastroenteritis. We developed a sensitive and rapid assay using the murine hybridoma Ped-2E9 cell model for the detection of these toxins. Bacillus culture supernatants containing toxins were added to the Ped-2E9 cell line and analyzed for cytotoxicity with an alkaline phosphatase release assay. Data were comparable to those obtained with the standard Chinese hamster ovary (CHO)-based cytotoxicity assay, which took about 72 h to complete. The Ped-2E9 cell assay had 25- to 58-fold-higher sensitivity than the CHO assay. Enterotoxin-producing Bacillus thuringiensis also gave positive results with Ped-2E9 cells, while several other Bacillus species were negative. Toxin fractions of >30 kDa showed the highest degree of cytotoxicity effects, and heat treatment significantly reduced the toxin activity, indicating the involvement of a heat-labile high-molecular weight component in Ped-2E9 cytotoxicity. This Ped-2E9 cell assay could be used as a rapid (1-h) alternative to current methods for sensitive detection of enterotoxins from Bacillus species. Currently we are analyzing the toxin component responsible for cytotoxicity observed in Ped-2E9 and CHO cells by HPLC and Mass Spectrometric analysis. Mechanism of intestinal phase of Listeria monocytogenes 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 and it participates in Listerial attachment to intestinal cells. LAP mediated adhesion appears to be primarily involved in the lower part of small intestine (ileum) and the upper part of large intestine (cecum and colon). LAP is mostly cytosolic and is determined to be alcohol acetaldehydrogenase encoded by the aad gene. The expression of aad is thermoregulated and nutrient-rich environment or high glucose down regulates, while nutrient -limiting environment up-regulate expression. The aad gene is located outside the virulence gene cluster and is not regulated by PrfA. In mouse feeding trial, aad-deficient mutant strains had significantly reduced adhesion, invasion and translocation to spleen, liver and brain tissues than the WT. The receptor for LAP was identified from mammalian cells to be heat shock protein 60 (Hsp60). Hsp60 is a chaperon protein and its surface expression is found in select mammalian cells where L. monocytogenes binding is found to be higher. 
Text Box: Bhunia Lab

 Hyochin (foreground) and Dr. Oak  developing a multi-pathogen biosensor system.

(L) Dr. Amornrat (foreground) and Amanda (back) working on  BARDOT and (R) Light Scatterometer (BARDOT)

Kristin working on  the surface expression of Hsp60 in intestinal cell lines