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Food Safety Presentations

Bioprocess Modeling of Fouling Phenomena in Cross-flow Microfiltration of Viable Bacteria

X. Li, X. Ku, T. Kreke, K. Foster, E. Ximenes, J. Hardenstein, X. Liu, M. Ladisch, 251st National ACS Meeting, Biofuel & Biobased Chemical Production: Biomass Pretreatment and Hydrolysis, San Diego, CA, March 14, 2016, , 


Abstract: The detection and characterization of bacterial food pathogens from homogenates of meats and vegetables will benefit from methods that accelerate their concentration and recovery. We report an approach based on hollow fiber membrane microfiltration that enables small volumes of viable bacteria to be rapidly concentrated from a large volume of food extract. The resulting sample is in a form amenable to probing for presence of pathogens using PCR or antibody reagents. However, fouling at the membrane surface must be addressed since the bacteria are present in a background of proteins, colloidal particles, and macromolecules which can accumulate within, or at the surface, of the membrane pores. Otherwise, this becomes a major impediment to achieving sustainable fluxes across membranes even though the porosity, equivalent to a 0.2 to 0.45 micrometer cutoff, is relatively large. This paper presents a bioprocess model of microfluidic transport that describes transmembrane pressure, flux, and deposition of a fouling layer as a function of distance from the entrance of a hollow fiber membrane during crossflow filtration of aqueous protein homogenates. Applications of the model in identifying optima for the membrane's geometric configuration will be discussed in the context of an approach that combines enzyme pretreatment of the initial sample followed by pre-filtration and crossflow microfiltration. The model identifies conditions that control membrane fouling so that efficient and reproducible concentration and recovery of bacterial cells in a viable form is achieved. Wider application of this model to microfiltration of other biological media will also be presented.

Research Area: Food Safety

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Food Pathogen Concentration, Recovery and Detection by Combined Use of C3D and CDx Technology

X. Liu, J. Hardenstein, S. Ku, T. Kreke, K. Foster, K. Jeffries, E. Ximenes, M. Ladisch, 17th Annual USDA-CFSE Meeting, Purdue University, November 16-17, 2015, , 


Abstract: Application of hollow fiber technology to food pathogen concentration and recovery has significant potential for reducing the time required to detect contamination in food. The Laboratory of Renewable Resources Engineering (LORRE) has designed and developed the Continuous Cell Concentration Device (C3D), which utilizes cross-flow microfiltration to rapidly separate and concentrate pathogens from liquid samples. The automated device consists of a 0.2um hollow fiber membrane module and two peristaltic pumps to recirculate flow, achieving large sample volume reduction and concentration of pathogenic populations such as E. coli O157:H7, Salmonella and Listeria. With a large hollow fiber surface area available for filtration, flow rates are increased for solutions with high-protein content, such as beef or chicken homogenates. However, microfiltration of food solution poses challenges due to its complex matrix of fat, proteins, colloids, and other macromolecules. To enable microfiltration, food solutions are usually pre-treated with enzyme and pre-filtered prior to C3D processing. Food solutions are then concentrated in the C3D and probed for potential pathogenic populations. Previous experimental results show 68% recovery of E. coli in ground beef and 70-80% recovery of Salmonella in chicken homogenates. This research aims at adapting a novel technology for detection of E. coli in post-processed C3D samples. Crystal Diagnostics' Xpress (CDx) system utilizes a liquid crystal biosensor for detection of E. coli. Antibodies added during sample preparation form microbial aggregates, which when placed on a BioCassette, distort the aligned liquid crystal matrix, enabling detection. With this system, both higher (107-108 CFU/mL) and lower (105-106) concentrations were detected.

Research Area: Food Safety

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Bioseparation and Analysis Techniques for Food Safety Inspection

M. R. Ladisch, E. Ximenes, T. Kreke, K. Foster, S. Ku, L. Liu, A. Deering, 16th Beijing Conference and Exhibition on Instrumental Analysis, Session G: Analytical Techniques in Life Sciences, Beijing, China, October 29, 2015 , , 


Abstract: The detection of pathogenic microorganisms in foods is an important component of food safety. The requirements for pathogen detection include obtaining a representative sample of the food being tested, and then amplifying or concentrating the microorganisms present so that the viable cell count is high enough to enable detection of pathogens, if present. The time that elapses between sampling and detection is preferably less than 8 hours, so that the result may be achieved within one work shift. Methods that address these goals will be addressed and include cross-flow microfiltration and enrichment culture coupled to PCR. The recovery and concentration of microorganisms from various types of foods, and the detection of Salmonella for purposes of food safety inspection will be discussed.

Research Area: Food Safety

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Enzyme-Assisted Pathogen Detection Applied to a Microfiltration System for Food Safety

Jaycey Hardenstein, Alisha Tungare, Xingya Liu, Eduardo Ximenes, Michael Ladisch, presented at Posters on the Hill, Washington, DC, April, 2015, , 


Abstract: With a growing number of consumers in the American market and with food production at an all-time high, food safety is a huge priority for both consumers and corporations everywhere. Recently, the Laboratory of Renewable Resources Engineering (LORRE) at Purdue University, developed a Continuous Cell Concentration Device (C3D) that has the potential to reduce the amount of time required to detect foodborne pathogens. The C3D utilizes microfiltration to produce a smaller, concentrated sample, which facilitates the identification of microbial populations. Before cell concentration, food samples are subjected to a pretreatment process that utilizes enzymes to prevent the build-up of proteins and large molecules that can plug the hollow fibers used in the C3D. Pretreated samples are then run through the C3D to recover a solution with a higher concentration of microbial cells. Our research investigates the role of enzymes to enable microfiltration and ensure recovery of Escherichia coli (E. coli) in ground beef solutions. We are working to quantify the effect of enzyme pretreatment E. coli cell viability. Experiments are currently being conducted to determine the effect of enzyme treatment, if any, on microbial cell growth and to optimize the amount of enzyme used. Preliminary results show that enzyme pretreatment effectively breaks down large proteins and prevents fouling of the membrane, as enzyme-treated solutions filter four times faster than untreated food solutions and recover more than 90% of E. coli during the pretreatment process. Thus, enzyme pretreatment, coupled with C3D technology, begins to address the critical need for rapid pathogen detection.

Research Area: Food Safety

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FDA Food Safety Challenge

M. Ladisch, E. Ximenes, K. Foster, A. Deering, T. Kreke, X. Liu, Seockmo Ku, 5th Annual FDA Foods and Veterinary Medicine Science and Research Conference, Silver Spring, MD, August 13, 2015, , 


Abstract: The Centers for Disease Control and Prevention (CDC) report that viruses are major causative agents for foodborne illnesses, although the most severe cases are associated with bacteria including Salmonella species. Salmonella (non-typhoidal) and Toxoplasma gondii are the first and second most costly foodborne pathogens in the United States. Our concept addresses concentration, recovery and detection of pathogens, specifically Salmonella, starting with stomaching of food sample followed by pre-filtering through glass microfiber (2.7 um) or nylon (10 um) membranes to remove larger particulates. Enzyme is added to neutralize agents in the extract that foul microfiltration membranes. Next, cross-flow microfiltration with a commercial polyethersulfone hollow fiber membrane module (0.2 um cut-off) removes liquid and retains microorganisms in viable and concentrated form. The microfiltration module is an integrated component of an automated instrument, developed in our laboratory, for accelerating sample preparation to detect Salmonella in unprepared foods. The type of food determines which enzymes are selected to remove fouling agents. Cellulases, hemicellulases and pectinases are used for vegetables and fruits, while proteases are key enzymes for meat and egg samples. Since maintaining viability of the microorganisms is critical, we have selected and tested enzymes effective at conditions that maintain viability. Microfiltration of enzyme-treated extract is based on an automated Continuous Cell Concentration Device (C3D). This system is the result of laboratory research with a series of prototypes that were successively designed, constructed, tested and improved to validate materials of construction, operability, cleaning (sterility) cycles, automation, control of membrane fouling, and recovery of viable microorganisms by quickly processing a large sample volume into a small one. The C3D carries out automated, cross flow microfiltration of up to 500 mL of food extracts into a 0.5 to 2 mL sample containing viable microbial cells in a concentrated form. A short enrichment step (using selective medium such as Rappaport Vassiliadis (RV) broth for Salmonella) further increases cell numbers by 10%, and is particularly useful for a low initial number of pathogens (less than or equal to 1 CFU/g) and/or reduction of non-target naturally-occurring microorganisms. Recoveries of target microorganisms range from 50 to 100% in 0.5 to 2 mL sample volumes obtained from 50 to 500 mL extract. The entire process, including a short enrichment step of 1 hr and PCR analysis is completed in 8 hours. Sample handling, preparation, and instrument sterilization corresponds to an elapsed time of 3 hr. Subsequent concentration through C3D requires between 15 min and 1.5 hr, depending on the type and size of sample volume being processed. Detection through PCR adds 2 hrs. If a pathogen is detected, confirmation occurs by the next day by plating concentrate on selective medium. The system and the hollow fiber membranes are cleaned and sterilized for re-use through sequential application of sodium hydroxide, water, ethanol, and water. This procedure enables the microfiltration membranes to be re-used 15 times or more. Target pathogens are Salmonella sp and Listeria monocytogenes.

Research Area: Food Safety

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Optimization of Pretreatment Steps Applied to a Microfiltration System for Rapid Pathogen Detection

Hardenstein, Jaycey, Tungare, Alisha, Ladisch, Michael, Liu, Xingya, and Ximenes, Eduardo, , 


Abstract: With a growing number of consumers in the American market and with food production at an all-time high, food safety is a huge priority for both consumers and corporations everywhere. Recently, the Laboratory of Renewable Resources Engineering (LORRE), at Purdue University, developed a Continuous Cell Concentration Device (C3D) that has the potential to reduce the time required to detect food pathogens. In LORRE's research, food samples are subjected to enzyme pretreatment and pre-filtration to prevent protein aggregation and the subsequent plugging of hollow fiber membranes used in the C3D microfiltration process. The pretreated food samples can then be run through the C3D to recover a concentrated cell solution. Our research investigates the role of pre-filter materials and enzymes to enable microfiltration and ensure the recovery of non-pathogenic filter materials and enzymes to enable microfiltration and ensure the recovery of non-pathogenic Escherichia coli bacterial cells. The ideal pre-filter material would allow for a large cell recovery while also removing enough particles so that the sample will not plug the C3D. It was determined that the most effective pre-filter material for turkey extract samples was the Advantec 101 filter paper. Through quantifying the reduction of E. coli colonies, the Advantec 101 filter paper recovered 80-90% of cells. On the other hand, the GF/D filter currently used in the pre-filtration process resulted in only 40-60% bacterial cell recovery. In addition, experiments are currently being conducted to discover how enzyme treatment affects the characteristics of ground beef extract solutions, such as: pre-filtration speed, cell concentration time, and E. coli cell recovery. Ultimately, this research begins to address the critical need for rapid pathogen detection.

Research Area: Food Safety

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Rapid Sample Processing for Pathogen Detection

M. Ladisch, E. Ximenes, H. Vibbert, L. Liu, A. Bhunia, R. Bashir, J. Shin, and R. Linton, AIMBE 20th Annual Event, Washington, DC, February 21, 2011


Abstract: The rapid sample processing of extracts from food matrices is an essential component for rapid detection of food pathogens and food safety. The objective of our research is to develop and integrate operational technologies that rapidly and effectively concentrate viable target cell from food matrices and to couple concentration with interrogation for the presence of pathogens. Rapid detection requires rapid sample concentration and amplification of the target population, interrogation of a concentrated sample of cells containing both non-pathogenic and pathogenic organisms, and identification of the type of pathogenic organism should a target population such as Salmonella sp., Listeria sp., or E. coli be detected. This work shows the development of an automated instrument to concentrate and recover cells from both natural flora and artificially spiked organisms from foods is possible using membrane separations. This work describes the properties of food extracts containing microbial cells with respect to fouling of membranes as well as methods that overcome the fouling issues so rapid concentration in less than 30 min may be achieved. A combination of pre- and post-filtration protocols are required so that the instrument itself can be automated, and membrane filtration devices cycled through repeated uses. The utility of this approach has been demonstrated with microorganisms recovered from food samples (specifically chicken rinse), where 500 x in less than 60 min.

Research Area: Food Safety

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New Method for Fast Detection of Improved Degradability in Genetically Modified Plants

E. A. Ximenes, Y. Kim, X. Li, H. Vibbert, P. Rubinelli, N. Bonawitz, R. Meilan, C. Chapple, and M. R. Ladisch,  31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May 3-6, 2009


Abstract: Plant genetic engineering is considered a potential approach to reduce costs for biofuel production from lignocellulosic material. However, the ability to control cell-wall composition without compromising plant performance jis a key objective of bioenergy crop improvements. Plants have been engineered for the production of enzymes within the crop biomass, with an aim to minimize the costs of catalyst production in bioreactors. Future research on the upregulation of cellulose and hemicellulose biosynthesis pathway enzymes for an increase in polysaccharides may also have the potential to improve cellullosic feedstocks. The most successful efforts to date have focused on the modification of lignin quantity and/or quality, in an effort to obviate the need for expensive pretreatment processes. Here we report a method for rapid detection of improved biodegradability in genetically modified plants that vary in lignin content and/or composition. For this purpose, only 50 mg of ground material is needed for liquid hot water pretreatment, and the method allows the pretreatment of up to 9 samples every 10 min per sandbath. Enzyme hydrolysis in the presence of commercial cellulases and beta-glucosidase is performed in a final volume of 1 mL for 30 min, at 50 C, and pH 4.8. The samples are then centrifuged, and the amount of glucose liberated is analyzed via a microplate assay. Using this approach, we have been able to rapidly and reproducibly identify genetically modified plants jwith improved biodegradability.

Research Area: Food Safety

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Separations Challenges for Aqueous Separations

M. R. Ladisch, R. Bashir, A. Bhunia, Y. Kim, and N. Mosier, 2008 Annual Meeting of the American Institute of Chemical Engineers, Philadelphia, PA, Plenary Session I on Bioseparations: Celebrating 100 Years of Bioseparations, November 17, 2008


Abstract:

Research Area: Food Safety

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Fundamentals of Nanotechnology in Agriculture

Ladisch, M. R., T. Huang, R. Armstrong, and N. Mosier, Division of Agricultural and Food Chemistry, Paper 205, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 17, 2005


Abstract: Nanoscience is the fabrication, study, and modeling of principles of devices and structures for which at least one dimension is several 100 nanometers or smaller. Nanotechnology is the enabling component of the discovery and development process that assembles nano-structures into compact, portable devices that carry out sensing functions currently relgated to scientific laboratories. Some types of devices will integrate biotechnology with silicon or plastic surfaces to form biosensing systems that enhance detection and enable study of biomarkers generated in resonse to environmental stress and other biological conditions of importance to agriculture. When coupled with devices that have capabilities to give temporal and geograhic information, nanotechnology may contribute to tracking of agricultural commodities. This paper will discuss possible applications of very small, intelligent, sensing devices for monitoring products from a widely distributed, global agricultural enterprise, and their potential contribution to identify preservation.

Research Area: Food Safety

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Nanoscale, Enzyme Mimicking Catalysts for Bioprocessing Agricultural Residues

Mosier, N. S., Division of Agricultural and Food Chemistry, 229th ACS Annual Meeting, San Diego, CA, March 16, 2005


Abstract: Developments in the understanding of the nanoscale structure and molecular mechanism of cellulolytic enzymes provide insights that may guide the development of nanoscale catalysts that efficiently hydrolyzes cellulose and hemicellulose from agriculturally derived feedstocks. Nanoscale, biomimetic catalysts may provide cost effective means for producing fermentable sugars from lignocellulosic biomass for renewable fuel and chemical production from agriculturally derived jplant biomass. This enzyme mimetic is composed of two functional domains: a catalytic domain and a cellulose binding domain. The cellulose binding domain selectively adsorbs the acid catalytic domain to the cellulose surface, thus concentrating the catalyst at the substrate surface. Maleic acid, a leading catalytic domain, effectively hydrolyzes cellulose with the glucose degradation when compared against mineral acids such as sulfuric acid. Maleic acid was found to be capable of yielding at least 50% more fermentable glucose from microcrystalline cellulose and corn stover compared to sulfuric acid at similar acid strength and hydrolysis conditions. When coupled with a cellulose binding domain, maleic acid may be concentrated near the cellulose surface. A number of cellulose binding domain candidates have been screened for adsroption to cellulose at hydrolysis conditions (>100 C, > 1 atm). Effective binding domain candidates have physiochemical properties similar to enzyme binding domains - planar, hydrophobic molecules capable of hydrogen bonding. Indole, the side chain of the amino acid tryptophan which is critical for enzymatic adsorption, has been showed to adsorb to cellulose at these conditions.

Research Area: Food Safety

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Nanotechnology and Press-fit Microdevices

Huang, T., D. Taylor, N. S. Mosier, M. Sedlak, and M. R. Ladisch, Novel Applications: Nanobiotechnology, 2nd World Congress on Industrial Biotechnology and Bioprocessing, Orlando, Florida, April 20, 2005


Abstract:

Research Area: Food Safety

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Predicting Cell Capture from Dilute Samples for Microfluidic Biosensors

Mosier, N. S., Craig, B.,  Workshop on Overarching Issues in Risk Analysis, National Institute of Statistical Sciences, Ames, IA, October 28, 2005


Abstract:

Research Area: Food Safety

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Press-fit Microdevice for Detecting Pathogenic Bacteria

Huang, T., D. G. Taylor, X. Liu, M. Sedlak, N. S. Mosier, and M. R. Ladisch, Poster, Indiana Biosensor Conference, Indianapolis, Indiana, April 6, 2005


Abstract: We report a rapid microfluidic device construction technique which does not employ lithography or stamping methods. Device assembly physically combines a silicon wafer, an elastomer (polydimethylsiloxane (PDMS)), and microfibers to form patterns of hydrophobic channels, wells, elbows, or orifices that direct fluid flow into controlled boundary layers. Tweezers are used to place glass microfibers ina defined pattern onto an elastomeric (PDMS) hydrophobic film. The film is then manually pressed onto a hydrophobic silicon wafer causing it to adhere to the silicon wafer and form a liquid-tight seal around the fibers. Completed in 15 minutes, the technique results in an operable microdevice with micron scale features of nanoliter volume. Microfiber-directed boundary flow is achieved by usse of the surface wetting properties of the hydrophilic glass fiber and the hydrophobicity of surrounding surfaces. The simplicity of this technique allows quick prototyping of microfluidic components, as well as complete biosensor systems, such as we describe for the detection of pathogenic bacteria. E. coli cells that express green fluorescent protein (GFP) or mixtures of non-pathogenic and heat-killed E. coli O157:H7 cells incubated and labeled with fluorescein-conjugated antibodies were readily detected and counted with this device.

Research Area: Food Safety

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Probability Distribution Model for Predicting Cell Capture from Dilute Solutions for Microfluidic Biosensors

Zang, Y., X. Liu, B. Tyner, A. Stewart, W.-T. Chen, M. Sedlak, N. S. Mosier, B. Craig, and M. R. Ladisch, Division of Biochemical Technology, Poster 341, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 16, 2005


Abstract: The detection of low numbers of organisms in large volumes of liquids is a challenge for both the fermentation and food industries. The detection of microbial contamination or the presence of pathogens requires that the sample be processed, concentrated, and assayed to detect living cels. The rapid concentration and detection of the pathogen, Listeria monocytogenes, from liquid extract of meat is one application where sampling size to achieve adequate detection confidence levels is crucial. The prediction of the minimal sample volume required to enable detection of a specified microorganism must be carefully carried out so that the probability of detection meets pre-determined criteria. We show that detection of 10 to 50 living cells extracted from a 50 g meat sample into 250 mL of buffer can be calculated using the Poisson distribution equation. Using GFP expressing E. coli that can be individually visualized microscopically, we show that a random distribution model accurately represents the probability of detection as a function of sample volume and concentration. This work is generalized to the detection of bacteria in meat, vegetable, and fermentation broth. The significance of these results in the context of rapid detection of pathogens using microfluidic devices for purposes of bioprocess monitoring and control is discussed.

Research Area: Food Safety

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Detection of Labeled Microbial Cells Using Microfluidic Biosensor

Huang, T., D. Taylor, M. Sedlak, G. Gregori, D. Akin, R. Bashir, M. R. Ladisch, and P. Robinson, BIOT Division, Paper 10, 227th ACS National Meeting, Section: Biosensors and Bioprocess Monitoring and Control, Anaheim, CA, March 28, 2004


Abstract: We demonstrate a simple and rapid (1 hour) technique for fabricating microfluidic flow channels using microfibers positioned on a galss or silica surface, and covered with a preformed poly(dimethylsiloxane) (PDMS) that binds to the surface to give a liquid seal. We used this technique to construct hydrohobic microchannels with a microfiber at its center. This design allows a 5 micron wide stream of liquid to be focused along the side of the microfiber. This phenomenon, utilized in combination with a conventional epi-fluorescence microscope and a photometer allows us to count fluorescently labeled bacteria. A model that quantitates both bacterial motility and convective motion due to fluid movement predicts movement of cells in this microfluidic device. Application of the model, combined with the facile assembly of microfluidic channels, enables biosensors to be designed that integrate microfluidic transport, separation, and detection of pathogenic and non-pathogenic microbes.

Research Area: Food Safety

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Pathogen Capture and Concentration on Functionalized Polycarbonate Membrane Detection and Sample Preparation Based on Immuno-Filters

Chen, W.-T. (Speaker), M. R. Ladisch, T. Geng, and A. K. Bhunia, BIOT Division Paper 140, 227th ACS National Meeting, Section: High Throughput Screening/Genomics and Proteomics, Anaheim, CA, March 30, 2004


Abstract: Rapid concentration and recovery of bacterial cells for the purpose of pathogen detection fluid, derived from meat, may be achieved in less than 30 min using polycarbonate membranes. Concentration of microbial cells accompanied by selective capture of a pathogenic microbe, such as Listeria monocytogenes, from non-pathogenic E. coli, requires a membrane that is functionalized with an antibody capable of selectively capturing the target microbe. We report immobilization techniques that enable attachment of an antibody that is specific to our target organisms, L. monocytogenes, based on Poly-L-Lysine activation of the membrane surface. Subsequent immobilization of the antibody in the presence of glutaraldehyde resulted in a functionalized membrane surface for selective capture of L. monocytogenes from a mixture containing E. coli. The selectivity of the membrane is demonstrated using both imaging and culture techniques. Retention characteristics are modeled based on equilibrium binding of microbe to membrane.

Research Area: Food Safety

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A Novel Method to Detect Listeria monocytogenes with Bio-selective Membranes

Chen, W.-T., (Poster presenter), M. R. Ladisch, T. Geng, and A. K. Bhunia, BMES Annual Meeting, Nashville, TN, October 2003


Abstract: Membrane filtration has been used widely in separation processes for a long time. It has the ability to sort out different substances based on size difference and also concentrate the target into a smaller area. Our previous study showed success in using polycarbonate membrane (PC) to recover our target organism Listeria monocytogenes, it gives us ideas how we can expand this membrane from simple separation tool to detection platform where separation and pathogen detection can be done simultaneously. PC has defined pore size and pore pathway has made PC a very good candidate for screen filter and our antibody immobilization. The microorganism of interest is Listeria monocytogenes. It is detrimental for immunocompromised people, like the elderly, infants and pregnant women. The average death rate is ~28%, but can be as high as 70% for these immunocompromised persons. L. monocytogenes usually occurs in ready-to-eat (RTE) dairy food, such as hotdogs, cheese and milk. All the facts above have led the U.S. Department of Agriculture (USDA) to set up a "zero tolerance" for L. monocytogenes.

Research Area: Food Safety

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Bio-mediated Assembly of Functionalized Microbeads for Capture of Microorganisms

Huang, T., T. Geng, D. Akin, W. Chang, J. Sturgis, R. Bashir, A. K. Bhunia, J. P. Robinson, and M. R. Ladisch, BIOT Division Paper 97, 225th ACS National Meeting, Section: Novel Bioanalyses Using Lab-on-a-Chip Technologies, New Orleans, LA, March 24, 2003


Abstract: There has been a growing interest to combine microbeads-based surfaces with microfluidic devices to provide bead-based surfaces with microfluidic devices to provide bead-based separation, detection or analysis of specific biological species. This paper reports fabrication of functionalized particulate monolayer on a C18 coated SiO2 surface via bio-mediate self-assembly or adsorption. A microchip with C18 surface pre-adsorbed with biotinylated BSA enables rapid self-assembly of streptavidin coated microbeads through specific biotin-streptavidin interaction. When coated with BSA, this microchip surface immobilizes polystyrene or dimethylamino beads through possible non-specific hydrophobic or electrostatic interactions. Protein coated microbeads such as ones coated with anti-Listeria antibody cana lso be immobilized onto a bare C18 surface through hyrophobic interactions. A microbead patterned surface results where the only area capable of binding proteins or microbes is the microbead itself, since biotinylated BSA or BSA pre-adsorbed onto a C18 surface blocks non-specific adsorptions. Fluorescnece microscopy was used in this work to study the adsorption of Escherichia coli and the food pathogen Listeria monocytogenes onto various types of microbeads immobilized on microchip surfaces. Four types of microbead coated surfaces were used: (1) streptavidin microbeads surface pre-adsorbed with biotinylated anti-Listeria antibody; (2) anti-Listeria microbead surfaces pre-blocked with BSA; (3) polystyrene microbead surfaces; and (4) dimethylamino microbead surfaces. Streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody coated microbeads showed specific capture of L. monocytogenes while polystyrene microbeads and dimethylamino microbeads captured E. coli and L. monocytogenes non-specifically. The combined use of functionalized microbeads for specific capture and biotinylated BSA or BSA for blocking non-specific adsorption enables development of fully functional microfluidic devices for separation, detection or analysis of specific biological species.

Research Area: Food Safety

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Biofunctional Membranes for Listeria monocytogenes Detection

Chen, W. T., M. R. Ladisch, T. Geng, and A. K. Bhunia, 225th ACS National Meeting, BIOT Division Paper 112, Section: Advances in Bioseparations, New Orleans, LA, March 25, 2003.,  225th ACS National Meeting, Section: Advances in Bioseparations, New Orleans, March 25, 2003


Abstract: Membrane filtration has offered the advantage of concentrating substances into small volumes or areas. In this study, we utilized polycarbonate membrane filters with defined pore sizes, and paths for filtering innoculated samples and carried out membrane-based detection for Listeria monocytogenes, which is a foodborne pathogen related to several food recals and listeriosis outbreaks. Membranes immobilized with specific antibodies to L. monocytogenes were used to filter innoculates samples. The chemistry utilizes direction reaction of a spacer with the membrane surface, followed by reaction with a bifunctional cross-linker, glutaraldehyde. Polyclonal anti-Listeria antibody was reacted and covalently bound on this surface. Tests with L. monocytogenes showed capture of this bacteria, which is reduced when the blocking agent BSA is added to the mix. Mechanisms for bacterial capture during microfiltration will be discussed.

Research Area: Food Safety

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Fabrication of Microfluidic Channels Using Microfibers with Poly(dimethylsiloxane)

Huang, T., W.-J. Chang, D. Akin, R. Gomez, R. Bashir, N. Mosier, and M. R. Ladisch, Poster Session: Advances in Biosensors and Biomedical Sensors I, Paper 193a, AIChE National Meeting, San Francisco Hilton & Towers, San Francisco, CA, November 17, 2003


Abstract:

Research Area: Food Safety

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Composite Surface for Capture of Listeria monocytogenes on a Protein Biochip

Huang, T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, and M. R. Ladisch, BIOT Division Paper 24, 224th ACS National Meeting, Section: Bioanalyses in the Micro to Nano-flow Regime, Boston, MA, August 18-22, 2002


Abstract: The design and fabrication of protein biochips requires characterization of blocking agents that minimize non-specific binding of proteins or organisms. Non-specific adsorption of E. coli, Listeria innocua, and Listeria monocytogenes is prevented by BSA or biotinylated BSA adsorbed on SiO2 surfaces of a biochip that had been modified with C18 coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Since streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins including antibodies. Henve, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfcing protein, avidin or streptavidin, which in turn anchors biotinylated antibody, which in our case is antibody C11E9, that binds Listeria spp. Non-specific adsorption of another bacterium, E. coli, is minimized due to the blocking action of the BSA. Derivatization of the chip's surfaces and preparation of protein coated chips for anchoring of antibodies is discussed.

Research Area: Food Safety

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Composite Surface for Protein Biochips

Huang, T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, and M. R. Ladisch, Poster 22, International Society for BioMEMS and Biomedical Nanotechnology (ISBBN) Meeting, Columbus, OH, September 6-8, 2002


Abstract: This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C18 coated SiO2 substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C18 surface was used to mediate the surface assembly of streptavidin coated microbeads (2.5 um), while a bare C18 surface was used to immobilize anti-Listeria antibody coated microbeads (2.5 um) through hydrophobic interactions. For a C18 surface pre-adsorbed wth bovine serum albumin, hydrophobic polystyrene microbeads (0.8 um) and positively charged dimethylamino microbeads (0.8 um) were allowed to be self-assembled onto the surface. A complete monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of E. coli and Listeria monocytogenes on these microbeads based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody coated microbeads showed specific capture of Listeria monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and Listeria monocytogenes non-specifically. The preparation of microbeads based surfaces for the construction of microfluidic devices for separation, detection or analysis of specific biological species is discussed.

Research Area: Food Safety

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Rapid Sample Preparation of Foodborne Pathogens by Membrane Filtration

Chen, W.-T., R. Hendrickson, and M. Ladisch, BioMEMS and Biomedical Nanotechnology, Poster 24, Columbus, OH, September 6-8, 2002


Abstract: Detection of foodborne pathogens requires that food samples have to be processed in order to remove interferring factors, including food particles, proteins and lipids, and concentration microorganisms that are to be probed for the presence of pathogens. Conventional involving culture stes may require up to 7 days. In the current study, membrane filtration is able to concentrate the foodborne pathogen, Listeria monocytogenes by a factor of 95 x, with 90% recovery of microorganisms by filtering 50 mL of food samples innoculated with Listeria monocytogenes using a syringe filter. Tween 20 was required to prevent irreversible adsorption of the microorganism to the membrane, due to hydrophobic interactions. Polycarbonate, cellulose, nylon and PVDF membranes were tested for their ability to retain Listeria monocytogenes and to separate proteins from microorganisms. The polycarbonate membrane filters with straight through, mono-radial pores were proved to be the most successful one. The results show that Listeria monocytogenes concentrated in this manner gives sufficient volume of sample for processing on a protein biochip where as little as 1 uL of sample is needed.

Research Area: Food Safety

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Rapid Separation and Concentration of Bacterial Pathogens in Liquid Food Samples

Chen, W.-T., (Speaker), R. Hendrickson, M. R. Ladisch, T. Geng, and A. K. Bhunia, BIOT Division Paper 77, 224th ACS National Meeting, Section: Advances in Bioseparations, Boston, MA, August 18-22, 2002


Abstract: Biochips offer the promise of quickly detecting foodborne pathogens with time to result of 3 hours or less if the time-consuming microbial enrichment of samples can be avoided. The work addresses the concentration of microbial cells using membrane filtration that can be carried out in 15 minutes. Samples collected from foods are chemically and biologically complex and contain proteins, lipids and fine particles that must be removed to avoid fouling the surface of the biochip. PVDF, nucleopore, nylon and cellulose membranes have been studied for separation of contaminating components and for concentration of cells. The pore size ranges of 0.22 um and 0.45 um were examined. The surface charge of the membrane was negative and did not absorb the cells, since the pH of the samples gave the cells a negative charge, as well. However, capture of the cells by the membrane was also observed due to the membrane structure.

Research Area: Food Safety

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Transport of Fluids Using Microwicks in Microfluidic Devices

Huang, T., J. Sturgis, R. Bashir, J. P. Robinson and M. R. Ladisch, Paper 340c, AIChE National Meeting, Indianapolis Convention Center, Indianapolis, IN, November 6, 2002


Abstract: Microwicks formed from a continuous strand of twisted threads of natural or synthetic fibers, such as cotton, silk, nylon or polyester, are capable of transporting nanoliter to microliter amounts of fluids. When dipped into a liquid, the microwick can draw the liquid through small flow channels formed bypatterns of fibers, by the action of capillary forces. This work presents the potential application of using microwick to transport one or more liquids into a microfluidic device. It was shown that microliter amounts of bovine serum albumin (BSA) solution can be transported using silk fibers in a matter of seconds. Microwicks formed by silk fibers were also shown to be able to transport bacterial cells (Listeria innocua) that were suspended in a carbonate bicarbonate buffer. By interfacing the silk fibers with a glass flow channel, a rapid and reliable introduction of bacterial cells (Listeria innocua) into the glass microchannel through the microwick was clearly demonstrated.

Research Area: Food Safety

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Fundamentals of Nanotechnology: Relationship to Food Science and Technology

Huang, T., W. Chen, T. Geng, R. Gomez, R. Bashir, A. Bhunia, and M. R. Ladisch, Invited Lecture, Paper 45-1, 2004 Institute of Food Technologies Annual Meeting, Symposium: Nanoscale Science, Engineering and Technology for Food Safety and Engineering ", Las Vegas, NV, July 14, 2004


Abstract: Nanoscience is the fabrication, study, and modeling of principles of devices and structures for which at least one dimension is several 100 nanometers or smaller. Nanotechnology is the enabling component of the discovery and development process that assembles nano-structures into compact, portable devices that carry out sensing functions currently relegated to scientific laboratories. Some types of devices will integrate biotechnology with silicon or plastic surfaces to form bio-sensing systems that enhance detection and enable study of biomarkers generated in response to environmental stress and other biological conditions of importance to agriculture. When coupled with devices that have capabilities to give temporal and geographic information, nanotechnology may contribute to tracking of agricultural commodities. This paper will discuss possible applications of very small, intelligent, sensing devices for monitoring products from a widely distributed, global agricultural enterprise, and their potential contribution to identity preservation.

Research Area: Food Safety

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