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Food Safety Publications

Accelerating Sample Preparation Through Enzyme-Assisted Microfiltration of Salmonella in Chicken Extract

2015

Authors: H. B. Vibbert, S. Ku, X. Li, X. Liu, E. Ximenes, T. Kreke, M. R. Ladisch
Journal: Biotechnology Progress, 31(6), 1551-1562 (2015).
Book Chapter:

Abstract: Microfiltration of chicken extracts has the potential to significantly decrease the time required to detect Salmonella, as long as the extract can be efficiently filtered and the pathogenic microorganisms kept in a viable state during this process. We present conditions that enable microfiltration by adding endopeptidase from Bacillus amyloliquefaciens to chicken extracts or chicken rinse, prior to microfiltration with fluid flow on both retentate and permeate sides of 0.2 um cutoff polysulfone and polyethersulfone hollow fiber membranes. After treatment with this protease, the distribution of micron, submicron, and nanometer particles in chicken extracts changes so that the size of the remaining particles corresponds to 0.4-1 um. Together with alteration of dissolved proteins, this change helps to explain how membrane fouling might be minimized because the potential foulants are significantly smaller or larger than the membrane pore size. At the same time, we found that the presence of protein protects Salmonella from protease action, thus maintaining cell viability. Concentration and recovery of 1-10 CFU Salmonella/mL from 400 mL chicken rinse is possible in less than 4 h, with the microfiltration step requiring less than 25 min at fluxes of 0.028-0.32 mL/cm2 min. The entire procedure - from sample processing to detection by polymerase chain reaction - is completed in 8 h.

Research Area: Food Safety    

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Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection

2014

Authors: I.-H. Cho, A. D. Radadia, K. Farrokhzad, E. Ximenes, E. Bae, A. K. Singh, H. Oliver, M. Ladisch, A. Bhunia, B. Applegate, L. Mauer, R. Bashir, J. Irudayaraj
Journal: Annual Review of Analytical Chemistry, 7, 65-88, 2014
Book Chapter:

Abstract: Despite continuing research efforts, timely and simple pathogen detection with a high degree of sensitivity and specificity remains an elusive goal. Given the recent explosion of sensor technologies, significant strides have been made in addressing the various nuances of this important global challenge that affects not only the food industry but also human health. In this review, we provide a summary of the various ongoing efforts in pathogen detection and sample preparation in areas related to Fourier transform infrared and Raman spectroscopy, light scattering, phage display, micro/nanodevices, and nanoparticle biosensor. We also discuss the advantages and potential limitations of the detection methods and suggest next steps for further consideration.

Research Area: Food Safety    

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Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration

2013

Authors: Xuan Li, Eduardo Ximenes, Mary Anne Roshni Amalaradjou, Hunter B. Vibbert, Kirk Foster, Jim Jones, Xingya Liu, Arun K. Bhunia, and Michael R. Ladisch
Journal: Applied and Environmental Microbiology, 79(22), 7048-7054, 2013
Book Chapter:

Abstract: This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples, and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nomimal pore size of 0.2 um constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of innoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 102 CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.

Research Area: Food Safety    

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Mechanistic Study of Membrane Concentration and Recovery of Listeria monocytogenes

2005

Authors: Wan-Tzu Chen, Richard L. Hendrickson, Chia-Ping Huang, Deb Sherman, Tao Geng, Arun K. Bhunia, Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 89, 3, 263-273 (2005)
Book Chapter:

Abstract: Detection of the foodborne pathogen Listeria monocytogenes requires that food samples be processed to remove proteins and lipids, concentrate microorganisms to a detectable concentration, and recover the concentrated cells in a small volume compatible with micron-scale biochips. Mechanistic considerations addressed in this research include the roles of membrane structure, pore size, and detergents in maximizing recovery of cells from a complex biological fluid. The fluid in this case was a food sample (hotdog extract) innoculated with L. monocytogenes. This study showed how membrane filtration using a syringe filter is able to concentrate L. monocytogenes by 95 x with up to 95% recovery of living microorganisms by concentrating 50 mL of food samplejinto a volume of 500 uL. Tween 20 was added to the sample to prevent irreversible adsorption of the microorganism to the membrane and thereby help to ensure high recovery. Comparison of polycarbonate, mixed cellulose, nylon, and PVDF membranes with 0.2 to 0.45 um pores showed the 0.2 um polycarbonate membrane with straight through, mono-radial pores gives the highest recovery of living microorganisms. The mixed cellulose, nylon, and PVDF membranes have a fibrous structure whose characteristic openings are much larger than their effective pore size cut-offs of 0.22 or 0.45 um. We define conditions for rapid membrane-based cell concentration and recovery that has the potential to supplant enrichment steps that require a day or more. This approach has the added benefit of facilitating examination of a large amount of fluid volume by reducing its volume to a range that is compatible with the microliter scales of biochip or other biosensor detection systems.

Research Area: Food Safety     

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Membrane for Selective Capture of the Microbial Pathogen Listeria monocytogenes

2005

Authors: Wan-Tzu Chen, Michael R. Ladisch, Tao Geong, Arun K. Bhunia
Journal: AIChE Journal, 51(12), 3305-3308, 2005
Book Chapter:

Abstract: Membrane filtration is widely used for sterile filtration, flowthrough immuno-filtration, virus removal, and microbiological analysis.1,2,3,4,5,6 Prior work has described membranes that retain one protein over others.7,8 The purpose of this work is to capture a target species of living cells from a mixed population of microorganisms being concentrated in the retentate of solution being pushed through a membrane. We report selective capture of one living microorganism from another by an antibody immobilized on a lysine-modified polycarbonate membrane. Capture of a relatively large microbial entity (L. monocytogenes at 1 m) by a 75 to 150 fold smaller ligand (the antibody) occurs with high efficiency, when a spacer (poly-Llysine) reacted to the membrane’s surface is derivatized with an antibody (P66) specific to Listeria. The results show that isolation of the food pathogen L. monocytogenes from E. coli in less than 2 h is possible.

Research Area: Food Safety    

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Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

2004

Authors: Geng, T., Morgan, M. T., and Bhunia, A. K.
Journal: Applied and Environmental Microbiology, 70, 10, 6138-6146 (2004)
Book Chapter:

Abstract: Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 x 103 CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 x 104 or 2.8 x 107 CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.

Research Area: Food Safety    

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Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells

2004

Authors: J. L. Wampler, K.-P. Kim, Z. Jaradat, and A. K. Bhunia
Journal: Infection and Immunity, 72, 2, 931-936 (2004)
Book Chapter:

Abstract: The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Research Area: Food Safety     

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Impedance Microbiology-on-a-Chip: Microfluidic Bioprocessor for Rapid Detection of Bacterial Metabolism

2004

Authors: R. Gomez-Sjoberg, D. T. Morisette, and R. Bashir
Journal: Journal of Microelectromechanical Systems, 1-8, (2004)
Book Chapter:

Abstract: Detection of a few live bacterial cells in many industrial or clinical samples is a very important technological problem. We have developed a microscale technique for concentrating bacterial cells from a dilute sample, by factors on the order of 10 to the 4th power to 10 to the 5th power, and detecting their metabolic activity by purely electrical means. The technique was implemented on a silicon-based microfluidic chip where the cells are concentrated and incubated in a chamber with a volume of 400 pl. Concentration and capture are obtained by the use of dielectrophoresis on the bacterial cells, and metabolism detection is achieved by means of impedance measurements of the medium in which the bacteria are incubated. Performing impedance-based detection at the microscale results in drastically reduced detection times for dilute bacterial samples, thanks to the ability to efficiently concentrate and capture the cells in an extremely small volume. Such concentration eliminates the need to amplify the bacterial population by long culture steps. This detection technique can be used for a wide variety of applications.

Research Area: Food Safety     

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Investigating Pathogen-Specific TLR Signaling of Innate Immune Cells for Biosensor Applications

2004

Authors: A. Lottes, H. Oh, H. HogenEsch, M. Ladisch, J. Hutchcroft, A. Rundell
Journal: 30th NE Bioengineering Conference, April 17-18, 2004, Western New England College
Book Chapter:

Abstract: The goal of this project is to develop a real-time cell-based biosensor using Toll-like receptors (TLRs) for pathogen detection. Existing biosensors rely on technologies that recognize only specific target analytes, requiring prior knowledge of the possible contaminating agents. Innate immune cells express TLRs that recognize conserved pathogen-associated molecular patterns on bacteria, viruses, parasites and fungi. Using TLRs as the receptor element in this biosensor will eliminate the need for a priori knowledge of the threat. At least 10 different members of the TLR family are expressed on cells of the innatejimmune system, each responding to different attributes of pathogenic organisms. Through flow cytometry, TLRs 2, 4 and 9 have been identified on THP-1 cells, and TLRs 2, 3, 4, 5 and 9 have been detected on J774 cells. Western blotting has identified Erk activation upon lipopolysacharide (LPS), E. coli and Poly(I):(C) exposure in J774 cells, and upon LPS and E. coli exposure in THP-1 cells. Cellular model systems are being developed to distinguish between bacteria and virus by selective stimulation of TLR3 and TLR5 (TLR3 specifically recognizes double-stranded viral RNA and TLR5 detects bacterial flagellin). A target application of this technology is point--of-care diagnostics. Real-time detection of viruses in nasal or throat swabs could help decrease the inappropriate use of antibiotics.

Research Area: Food Safety    

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Optimization of a Rapid Dot-Blot Immunoassay for Detection of Salmonella entrica serovar Enteritidis in Poultry Products and Environmental Samples

2004

Authors: Z. W. Jaradat, J. H. Bzikot, J. Zawistowski, A. K. Bhunia
Journal: Food Microbiology, 21, 761-769, (2004)
Book Chapter:

Abstract: An immunoassay was developed for the detection of Salmonella serovar enteritidis in poultry and environmental samples. This assay consisted of a two-step procedure that involved an enrichment step using whole egg homogenate (EH) as the enrichment medium and detection by a monoclonal antibody (MAb)-based dot-blot assay. Egg homogenate enriched Salmonella enteritidis was heated to 100 C for 10 min in the presence of cholic acid, a detergent, to liberate the lipopolysaccharide (LPS) antigen in gelled egg matrix. This was subsequently transferred onto a nitrocellulose membrane for detection with MAb 2F11. Several commercially available media were compared with egg homogenate for their relative ability to resuscitate and propagate Salmonella enterititis to detectable levels. Incubation in EH, trypticase soy broth (TSB), and lactose broth (LB) resulted in comparable levels of Salmonella Enteritidis as demonstrated by viable plate counts. Salmonella enteritidis grown in TSB exhibited the greatest visual intensity showing a positive test when tested by the dot-blot assay. Incubation time necessary to detect one cfu of Salmonella enteritidis was reduced from 20 to 10 h using TSB as the enrichment broth. Addition of ferrous sulphate or ferrioxamine E or cholic acid in the enrichment broth had negligible negative effects on the growth of Salmonella. Salmonella enteritidis when incubated with a mixture of naturally contaminated or artificially innoculated competitive micro-organisms in environmental samples at a ratio of 1:10 to the 2nd power, was able to reproduce to detectable numbers for the immunoassay. This method was able to detect all phage types (PT 1, 6, 7, 8, 13, 13a, 14b, 21 and 28) with unique ribopatterns. The results demonstrated that Salmonella enteritidis, when pre-enriched in a medium containing ferrous sulphate or cholic acid, could be readily detected in the presence of 100-fold higher competition of other microorganisms.

Research Area: Food Safety     

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Characterization and Application of a Listeria monocytogenes Reactive Monoclonal Antibody C11E9 in a Resonant Mirror Biosensor

2003

Authors: Amanda A. Lathrop, Ziad W. Jaradat, Tim Haley, Arun K. Bhunia
Journal: Journal of Immunological Methods, 281, 119-128, (2003)
Book Chapter:

Abstract: Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44–3.58 and for L. innocua 0.22–1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6–0.99) and low (0.18–0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 108 cells/ml; however, it showed binding (85–150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 Ag/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly ( p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10–20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite crossreaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.

Research Area: Food Safety     

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Composite Surface for Blocking Bacterial Adsorption on Protein Biochips

2003

Authors: Huang, T. T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, M. R. Ladisch
Journal: Biotechnology and Bioengineering, 81(5), 618-624, (2003)
Book Chapter:

Abstract: The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO2 surfaces of a biochip that had been modified with a C18 coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C18-derivatized SiO2 surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.

Research Area: Food Safety     

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Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments

2003

Authors: T. Geng, K. P. Kim, R. Gomez, D. M. Sherman, R. Bashir, M. R. Ladisch, A. K. Bhunia
Journal: Journal of Applied Microbiology, 95, 762-777 (2003)
Book Chapter:

Abstract: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments.

Research Area: Food Safety     

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Microfiber Assisted Fabrication of Microfluidic Channels Using Poly(dimethylsiloxane)

2003

Authors: Tom T. Huang, Woo-Jin Chang, Demir Akin, Rafael Gomez, Rashid Bashir, Nathan Mosier, Michael R. Ladisch
Journal: AIChE Journal, 49, 11, 2984-2987 (2003)
Book Chapter:

Abstract: A microfluidic device is typically formed using bulk silicon etching techniques on a silicon substrate (Kovacs et al., 1998). A photolithography step defines the desired pattern on the silicon substrate with photoresist. Etching using acids or gases, followed by a solvent and acid cleaning process to remove residual photoresist, leaves micron-scale features. Such devices are capable of providing rapid identification of nucleic acids, proteins, drugs, or other important biological compounds with enhanced sensitivity and time-to-result, while reducing consumption of expensive reagents compared to microtiter plates or test tube scale analyses (Stone and Kim, 2001; Khandurina and Guttman, 2002; Meldrum and Holl, 2002). Polydimethylsiloxane (PDMS), created by mixing a silicone elastomer base and a curing agent in a 10:1 ratio, gives an alternate material for fabricating microfluidic devices (McDonald and Whitesides, 2002). The liquid pre-polymer is poured over a master generated either from photolithography using a high resolution transparency as a photomask, or by laser ablation or Solid-Object Printing to form the device (Grzybowski et al., 1998; McDonald et al., 2002; McDonald and Whitesides, 2002). We report formation of a master by directed placement of glass microfibers on silicon or glass substrates, followed by pressing a preformed PDMS sheet onto the substrate to form microfluidic channels. Wells are formed by criss-cross fiber patterns, while functionalized microbeads coated onto fibers result in microscale channels that separate proteins. We believe this approach is an attractive research tool, because it places rapid prototyping capability within the reach of laboratories that have access to glass slides, an optical microscope, digital camera, tweezers, and PDMS.

Research Area: Food Safety     

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Pathogen Detection, Food-Borne

2003

Authors: R. Bashir
Journal: McGraw-Hill Yearbook of Science and Technology, 1-3 (2003)
Book Chapter:

Abstract: The presence of microorganisms in food is a natural and unavoidable occurrence. Cooking generally destroys most harmful bacteria, but undercooked foods, processed ready-to-eat foods, and minimally processed foods can contain harmful bacteria that are serious health threats. Meat, dairy, and poultry products are important reservoirs for many of the food-borne pathogens, including Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7. Animal by-products, such as feed supplements, may also transmit pathogens to food animals (for example, Salmonella and bovine spongiform encephalopathy). Seafood is another potential source of food-borne pathogens, such as Vibrio, Listeria, and Hepatitis A. Infectious doses of many of these pathogens are very low (~10 bacterial cells), increasing the vulnerability of the elderly, infants, and people with immunological deficiencies or organ transplants. Researchers are continuously searching for sensitive tools that are fast, accurate, and ultrasensitive. In recent years, there has been much research activity in the area of sensor development for detecting pathogenic microorganisms.

Research Area: Food Safety    

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Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip for Bacterial Culture

2003

Authors: Woo-Jin Chang, Demir Akin, Miroslav Sedlak, Michael R. Ladisch and Rashid Bashir
Journal: Biomedical Microdevices, 5, 4, 281-290 (2003)
Book Chapter:

Abstract: In this study, a novel PDMS/silicon hybrid microfluidic biochip was fabricated and tested for the long-term batch culture of bacterial cells. The PDMS (poly(dimethylsiloxane)) cover with 3-dimensional micro-channels for flow was fabricated using Teflon tubing and hole-punch techniques, without photolithographic methods. The PDMS/silicon hybrid biochip was prepared by bonding of PDMS cover and a silicon chip that had electrodes and micro-fluidic channels defined. The absorption of liquid into PDMS cover was characterized and conditions to prevent drying of nutrient media within the micro-chamber were shown. The absorption of liquid from micro-chambers into the PDMS cover was reduced up to 2.5 times by changing the mixing ratio of PDMS and curing agent from 10:1 to 2.5:1. In addition, pre-saturation of the PDMS cover with media prior to the incubation resulted in the preservation of liquid in the micro-chambers for up to 22 hours. Optimization of the mixing ratio and pre-saturation of the PDMS cover reduced the drying time 10 times when compared to the unsaturated PDMS cover composed of 10:1 ratio of PDMS and curing agent. Listeria innocua and a strain of Escherichia coli, expressing green fluorescent protein (GFP), were successfully cultured in batch mode within the PDMS/silicon hybrid biochip.

Research Area: Food Safety    

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Genetic Homogeneity Among Listeria monocytogenes Strains from Infected Patients and Meat Products from Two Geographic Locations Determined by Phenotyping, Ribotyping and PCR Analysis of Virulence Genes

2002

Authors: Jaradat, Z. W., G. E. Schutze, and A. K. Bhunia
Journal: International Journal of Food Microbiology, 76, 1-10, (2002)
Book Chapter:

Abstract: Thirty Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed for their genotypic and phenotypic virulence gene expressions and genetic relatedness. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB, with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically, all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups based on the similarity to DuPont Identification numbers (DID), which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are found in food or human hosts or are evenly distributed between these two. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinterR database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments. The isolates showed 92–99% genetic homogeneity, in spite of their origins from two different geographic locations and environments.

Research Area: Food Safety     

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Microfabricated Device for Impedance-Based Detection of Bacterial Metabolism

2002

Authors: Gomez, R., M. R. Ladisch, A. K. Bhunia, and R. Bashir
Journal: Materials Research Society Symposium Proceedings, 729 (2002)
Book Chapter:

Abstract: We present the use of a microfabricated device for impedance-based detection of a few live bacterial cells. Impedance-based detection relies on measuring changes in the AC impedance of two electrodes immersed in a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing bacterial cells. Rapid detection of a few cells (1 to 10) is possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to test this miniaturized assay. The conductance of the bacterial suspensions is extracted from measuring their complex impedance in a 5.27 nl chamber in the biochip, at several frequencies between 100 Hz and 1 MHz. Measurements on suspensions of the bacteria Listeria innocua, Listeria monocytogenes, and Escherichia coli in a low conductivity buffer demonstrate that, under the current experimental conditions, the minimum detection level is between 50 and 200 live cells, after two hours of off-chip incubation. Work is in progress to develop techniques for selective capture of bacteria inside the chip, and to minimize background changes in impedance during on-chip incubation.

Research Area: Food Safety     

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Microscale Electronic Detection of Bacterial Metabolism

2002

Authors: Rafael Gomez, Rashid Bashir, and Arun K. Bhunia
Journal: Sensors and Actuators B, 86, 198-208 (2002)
Book Chapter:

Abstract: In this paper, we present a microscale impedance-based technique for detecting the metabolic activity of a few live bacterial cells. Impedance-based detection relies on measuring changes in the ac impedance of two electrodes in contact with a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing cells. Rapid detection of a few live cells (1-10) is, in theory, possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to explore this technique, consisting of a network of channels and chambers etched in a crystalline silicon substrate. The complex impedance of bacterial suspensions is measured with interdigitated platinum electrodes in a 5.27 nl chamber in the biochip at frequencies between 100 Hz and 1 MHz. After 2 h of off-chip incubation, the minimum number of live cells suspended in a low conductivity buffer that could be easily distinguished from the same number of heat-killed cells was on the order of 100 Listeria innocua, 200 L. monocytogenes, and 40 Escherichia coli cells, confined into the 5.27 nl chamber. A number on the order of 100 live L. innocua cells suspended in Luria-Bertani (LB) broth produced a significantly higher signal than the same number of heat-killed cells, and a difference is evident even down to ~5-20 cells. To the best of our knowledge, this is the first demonstration of microscale impedance-based detection of bacterial metabolism.

Research Area: Food Safety     

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Adsorption of Avidin on Microfabricated Surfaces for Protein Biochip Applications

2001

Authors: Bashir, R., R. Gomez, A. Sarikaya, M. R. Ladisch, J. Sturgis, and J. P. Robinson
Journal: Biotechnology and Bioengineering, 73(4), 325-328 (2001)
Book Chapter:

Abstract: The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radio frequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.

Research Area: Food Safety     

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Impedance Spectroscopy and Biochip Sensor for Detection of Listeria monocytogenes

2001

Authors: Bhunia, A. K., Z. W. Jaradat, K. Naschansky, M. Shroyer, M. Morgan, R. Gomez, R. Bashir, and M. Ladisch
Journal: Proceedings of SPIE, (4206) 32-39 (2001)
Book Chapter:

Abstract: Listeria monocytogenes is a deadly foodborne human pathogen. Its ubiquitous nature and its ability to grow at refrigeration temperatures makes this organism a difficult one to control. High-volume processed, ready-to-eat (RTE) foods. Improved processing along with real-time detection could reduce the incidence of this pathogen. Conventional methods can detect this pathogen accurately, but take several days (2-7d) to complete, which is not practical considering the short shelf-life and cost fiber optic and microelectrical-mechanical system (MEMS) biochips were designed and examined for direct detection of L. monocytogenes from liquid samples. Also, interdigitated microsensor electrode (IME) chip and spectrofluorometer were used to measure L. monocytogenes interaction with mammalian cells (cytopathogenic activities) for indirect detection. Preliminary data generated using laboratory cultures of Listeria species indicated that L. monocytogenes could be detected in 30 min to 1 h 30 min depending on the techniques used.

Research Area: Food Safety     

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Microfluidic Biochip for Impedance Spectroscopy of Biological Species

2001

Authors: Gomez, R., R. Bashir, A. Sarikaya, M. Ladisch, J. Sturgis, J. P. Robinson, T. Geng, A. Buhnia, H. Apple, S. Wereley
Journal: Biomedical Microdevices, 3(3), 201-209, (2001).
Book Chapter:

Abstract: This paper describes the fabrication and characterization of a microelectronic device for the electrical interrogation and impedance spectroscopy of biological species. Key feature of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the nonplanar topography of the chip using spin-on-glass as an intermediate bonding layer. The total fluidic path volume in the device is on the order of 30 nl. Flow fields in the closed chip were mapped by particle image velocimetry. Electrical impedance measurements of suspensions of the live microorganism Listeria innocua injected into the chip demonstrate an easy method for detecting the viability of a few bacterial cells. By-products of the bacterial metabolism modify the ionic strength of a low conductivity suspension medium, significantly altering its electrical characteristics.

Research Area: Food Safety     

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