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Rapid Detection of Salmonella enteritidis in Pork Samples with Impedimetric Biosensor
Giyoung Kim,Ji Hea Moon,Bung-Kwon Hahm,Mark Morgan,Arun Bhunia,Ae Son Om 한국식품과학회 2009 Food Science and Biotechnology Vol.18 No.1
Frequent outbreaks of foodborne illness have been increasing the awareness of food safety. Conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Some immunological, rapid assays are developed, but these assays still require prolonged enrichment steps. Recently developed biosensors have shown potential for the rapid detection of foodborne pathogens. In this study, an impedimetric biosensor was developed for rapid detection of Salmonella entritidis in food sample. To develop the biosensor, an interdigitated microelectrode (IME) was fabricated by using a semiconductor fabrication process. Anti-Salmonella antibodies were immobilized based on neutravidin-biotin binding on the surface of the IME to form an active sensing layer. To evaluate the effect of electrode gap on sensitivity of the sensor, 3 types of sensors with different electrode gap sizes (2, 5, and 10 ㎛) were fabricated and tested. The impedimetric biosensor could detect 10³ CFU/㎖ of Salmonella in pork meat extract with an incubation time of 5 min. This method may provide a simple, rapid, and sensitive method to detect foodborne pathogens.
Yang, Liju,Banada, Padmapriya P.,Chatni, Mohammad R.,Seop Lim, Kwan,Bhunia, Arun K.,Ladisch, Michael,Bashir, Rashid Royal Society of Chemistry 2006 Lab on a chip Vol.6 No.7
<P>In this study, we demonstrated a micro-fluidic system with multiple functions, including concentration of bacteria using dielectrophoresis (DEP) and selective capture using antibody recognition, resulting in a high capture efficiency of bacterial cells. The device consisted of an array of oxide covered interdigitated electrodes on a flat silicon substrate and a ∼16 µm high and ∼260 µm wide micro-channel within a PDMS cover. For selective capture of <I>Listeria monocytogenes</I> from the samples, the channel surface was functionalized with a biotinylated BSA–streptavidin–biotinylated monoclonal antibody sandwich structure. Positive DEP (at 20 V<SUB>pp</SUB> and 1 MHz) was used to concentrate bacterial cells from the fluid flow. DEP could collect ∼90% of the cells in a continuous flow at a flow rate of 0.2 µl min<SUP>−1</SUP> into the micro-channel with concentration factors between 10<SUP>2</SUP>–10<SUP>3</SUP>, in sample volumes of 5–20 µl. A high flow rate of 0.6 µl min<SUP>−1</SUP> reduced the DEP capture efficiency to ∼65%. Positive DEP attracts cells to the edges of the electrodes where the field gradient is the highest. Cells concentrated by DEP were captured by the antibodies immobilized on the channel surface with efficiencies of 18 to 27% with bacterial cell numbers ranging from 10<SUP>1</SUP> to 10<SUP>3</SUP> cells. It was found that DEP operation in our experiments did not cause any irreversible damage to bacterial cells in terms of cell viability. In addition, increased antigen expression (antigens to C11E9 monoclonal antibody) on cell membranes was observed following the exposure to DEP.</P> <P>Graphic Abstract</P><P>Bacterial cells were concentrated by positive dielectrophoresis. Target cells were selectively captured by specific antibodies immobilized on the channel surface. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=b607061m'> </P>
Giyoung Kim,Mark Morgan,Daniel Ess,Byoung-Kwon Hahm,Aparna Kothapalli,Arun Bhunia 한국식품과학회 2007 Food Science and Biotechnology Vol.16 No.3
Conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Fiber-optic biosensors have been used to rapidly detect pathogens because they can be very sensitive and are simple to operate. However, many fiber-optic biosensors rely on manual sensor handling and the sandwich assay, which require more effort and are less sensitive. To increase the simplicity of operation and detection sensitivity, a binding inhibition assay method for detecting Listeria monocytogenes in food samples was developed using an automated, fiber-optic-based immunosensor; RAPTOR (Research International, Monroe, WA, USA). For the assay, fiber-optic biosensorswere developed by the immobilization of Listeria antibodies on polystyrene fiber waveguides through a biotin-avidin reaction. Developed fiber-optic biosensors were incorporated into the RAPTOR to evaluate the detection of L. monocytogenes in frankfurter samples. The binding inhibition method combined with RAPTOR was sensitive enough to detect L. monocytogenes (5.4ⅹ10? CFU/mL) in a frankfurter sample.
Binding Inhibition Assay Using Fiber-Optic Based Biosensor for the Detection of Foodborne Pathogens
Morgan, Mark T.,Kim, Gi Young,Ess, Daniel,Kothapalli, Aparna,Hahm, Byoung Kwon,Bhunia, Arun Trans Tech Publications, Ltd. 2006 Key Engineering Materials Vol.321 No.-
<P>Frequent outbreaks of foodborne illness have been increasing the need for simple, rapid and sensitive methods to detect foodborne pathogens. Conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Some immunological rapid assays are developed, but these assays still require prolonged enrichment steps. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Among the biosensors, fiber-optic methods have much potential because they can be very sensitive and simple to operate. Fiber-optic biosensors typically use a light transmittable, tapered fiber to send excitation laser light to the detection surface and receive emitted fluorescent light. The fluorescent light excited by an evanescent wave generated by the laser is quantitatively related to fluorophor-labeled biomolecules immobilized on the fiber surface. A portable and automated fiber-optic biosensor, RAPTOR (Research International, Monroe, WA), was used to detect Salmonella enteritidis in food samples. A binding inhibition assay based on the biosensor was developed to detect the bacteria in hot dog samples. The biosensor and the binding inhibition assay could detect 104 cfu/ml of bacteria in less than 10 min of assay time.</P>
Detection of Listeria Monocytogenes Using an Automated Fiber-Optic Biosensor: RAPTOR
Kim, Gi Young,Morgan, Mark T.,Ess, Daniel,Hahm, Byoung Kwon,Kothapalli, Aparna,Valadez, Angela,Bhunia, Arun Trans Tech Publications, Ltd. 2006 Key Engineering Materials Vol.321 No.-
<P>Fiber-optic biosensor uses light transmittable tapered fiber to send excitation laser light and receive emitted fluorescent light. The fluorescent light excited by an evanescent wave generated by the laser is quantitatively related to biomolecules immobilized on the fiber surface [1]. An automated fiber-optic biosensor based detection method for Listeria monocytogenes was developed in this research. Detections of Listeria monocytogenes in hotdog sample were performed to evaluate the method. By using the detection method with automated fiber-optic biosensor, 5.4×107 cfu/ml of Listeria monocytogenes was able to detect.</P>
Jagadeesan, Balamurugan,Koo, Ok Kyung,Kim, Kwang-Pyo,Burkholder, Kristin M.,Mishra, Krishna K.,Aroonnual, Amornrat,Bhunia, Arun K. Microbiology Society 2010 Microbiology Vol.156 No.9
<P>Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (lmo1634), interacts with host-cell receptor Hsp60 to promote bacterial adhesion during the intestinal phase of Listeria monocytogenes infection. The LAP homologue is present in pathogens (L. monocytogenes, L. ivanovii) and non-pathogens (L. innocua, L. welshimeri, L. seeligeri); however, its role in non-pathogens is unknown. Sequence analysis revealed 98 % amino acid similarity in LAP from all Listeria species. The N-terminus contains acetaldehyde dehydrogenase (ALDH) and the C-terminus an alcohol dehydrogenase (ADH). Recombinant LAP from L. monocytogenes, L. ivanovii, L. innocua and L. welshimeri exhibited ALDH and ADH activities, and displayed strong binding affinity (K(D) 2-31 nM) towards Hsp60. Flow cytometry, ELISA and immunoelectron microscopy revealed more surface-associated LAP in pathogens than non-pathogens. Pathogens exhibited significantly higher adhesion (P<0.05) to Caco-2 cells than non-pathogens; however, pretreatment of bacteria with Hsp60 caused 47-92 % reduction in adhesion only in pathogens. These data suggest that biochemical properties of LAP from pathogenic Listeria are similar to those of the protein from non-pathogens in many respects, such as substrate specificity, immunogenicity, and binding affinity to Hsp60. However, protein fractionation analysis of extracts from pathogenic and non-pathogenic Listeria species revealed that LAP was greatly reduced in intracellular and cell-surface protein fractions, and undetectable in the extracellular milieu of non-pathogens even though the lap transcript levels were similar for both. Furthermore, a LAP preparation from L. monocytogenes restored adhesion in a lap mutant (KB208) of L. monocytogenes but not in L. innocua, indicating possible lack of surface reassociation of LAP molecules in this bacterium. Taken together, these data suggest that LAP expression level, cell-surface localization, secretion and reassociation are responsible for LAP-mediated pathogenicity and possibly evolved to adapt to a parasitic life cycle in the host.</P>