The Center for Food Safety Engineeringg 2007 - 2008 Research Report

“Collaborating to make our food safer” The mission of the Center for Food Safety Engineering is to develop new knowledge, technologies and systems for detection and prevention of chemical and microbial contamination of foods.

Through CFSE, positions itself as a national leader in multi- disciplinary food safety research. Our multi- disciplinary approach, including a strong engineering component, makes Purdue University truly unique. 2007-2008 Research Report

2 Welcome from the Director • Message from Richard Linton, Center Director 2 Message from USDA • Message from our Partnership with USDA-ARS 3 Multipathogen screening using immunomicroarray • Arun Bhunia 4 Optical for food pathogen detection • Arun Bhunia 5 Optical forward scattering for bacterial colony differentiation and identifi cation • Arun Bhunia, E. Daniel. Hirleman, J. Paul Robinson 6 Immunocapture real-time PCR to detect mycotoxigenic mold spores in grains • Maribeth A. Cousin, Charles P. Woloshuk 7 Detection of foodborne pathogens via an integrated spectroscopy and -based approach • Joseph Irudayaraj, Lisa Mauer, Chitrita DebRoy, Pina Fratamico 8 Nanoparticle-based DNA-multiplexed probes for pathogen detection using confocal raman microscopy • Joseph Irudayaraj 9 Engineering of biosystems for the detection of Listeria monocytogenes in foods • Michael R. Ladisch, Rashid Bashir, Arun Bhunia, J. Paul Robinson 10 Spotlight on USDA-ARS Scientists 12 Rapid, quantitative, and reusable immunosensors for bacteria detection on a microfl uidic platform • Chang Lu, Arun Bhunia, Zhongyang Cheng 13 A method for capture and detection of Escherichia coli O157:H7 using polymer-immobilized phage • Mark Morgan, Bruce Applegate 14 Continuous monitoring of chemical agents in aqueous media using bioreporter-based sensors • David Nivens, Michael Franklin, Carlos Corvalan 15 Field-ready biosensors for high throughput and multiplexed detection of foodborne pathogens • Kinam Park, James Leary, Arthur Aronson 16 Portable biosensor for rapid and ultra-sensitive identifi cation of organophoshorous foodborne contaminants • Lia Stanciu, Silvana Andreescu 17 Scientifi c Publications and Presentations 20 Center Staff

Visit us @ www.cfse.purdue.edu Welcome from the Director

The year 2008 marks the eighth anniversary of the Center for Food Safety Engineering (CFSE) at Purdue University. Our partnership with the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) Eastern Regional Research Center continues to create signifi cant research and outreach impacts. This year, the CFSE team published 31 peer-reviewed research publications and presented 18 talks at national science meetings. CFSE scientists also have been forming partnerships with international countries. Scientifi c presentations and collaboration- seeking visits were made this year to Brazil, China, Hong Kong, and Scotland. Our trip to Jiao-Tong University in Shanghai, China was especially important in fostering a new international collaboration between USDA-ARS and Jiao-Tong University.

This year, our research program underwent a USDA-ARS Offi ce of Scientifi c Quality Review Dr. Richard H. LintoLintonn (OSQR), and the future direction of our research program was identifi ed. A research priority for DirectorDirector ofof the Center forfor the next few years is development of technological platforms that improve microbial and chemical Food SafetySafety EngineeringEngineering hazard detection. These new technologies will advance detection of bacterial pathogens including 765765.494.6481 494 6481 Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter spp., and Salmonella spp. and [email protected] of chemical hazards that may present food safety and food defense concerns. Our detection-based technologies will build upon prior success with optical biosensors, cell- based biosensors, bio-chips (lab-on-a-chip), microarrays, infrared spectroscopy (including Fourier transform infrared FTIR), enzyme linked immunosorbant assays, polymerase chain reactions, impedance-based microbiology, scanning microscopy, confocal raman microscopy, bioluminescence, DNA/RNA probes, and bioreporter-based chemical sensors. The exciting BActeria Rapid Detection using Optical scattering Technology (BARDOT) system is being evaluated by industry and regulatory agencies, and we hope that it will be available for wide-scale use sometime next year.

I continue to be impressed with the collaborative research efforts of Purdue University and USDA- ARS scientists and feel privileged to serve as director of the center. If you are interested in learning more about CFSE, please visit our Web site at www.cfse.purdue.edu or contact me directly.

Message from USDA

As the collaboration between the CFSE at Purdue University and the USDA-ARS Eastern Regional Research Center continues, I am grateful to witness the continual growth, maturation, and research impact of this partnership. We are pleased to learn that the ARS-Purdue team has successfully completed the USDA-ARS OSQR process with outstanding ratings. This result signals that the ARS-Purdue project is now considered to be an integral part of USDA-ARS research efforts. Together, we have received increased recognition as an important contributor to the technological advancement of pathogen detection in food—evidenced by the fact that the team received invitations from the International Workshop on Rapid Methods and Automation in Microbiology to conduct half-day symposiums on molecular methodologies in August 2006, June 2007, and June 2008. The team received extraordinary praise from Dr. Daniel Y. C. Fung, Professor of Food Microbiology, Kansas State University, for invaluable contributions to the workshop, and Dr. Shu-I Tu he extended an invitation for our continuing involvement in 2009. In May 2008, our team went to Supervisory Research Shanghai, China to attend the fi rst annual meeting of the Joint United States-China Food Safety Chemist USDA-ARS, Center, an international collaboration established between the ARS and Jiao-Tong University in Eastern Regional Shanghai. This collaboration is part of the cooperative research activities between the USDA and Research Center the Ministry of Science and Technology, China (MOST). With this collaboration in place, I believe 215.233.6466 that our ARS-Purdue team will become an important international research enterprise in the near [email protected] future.

2 Center for Food Safety Engineering Bhunia

Multipathogen screening using immunomicroarray Investigator: Arun Bhunia (Department of Food Science)

Project Rationale Project Objectives Antibody-based methods for detecting pathogenic foodborne • Develop a microarray assay in 96-well plate and bacteria are used widely and are regarded as rapid and effi cient. glass slide using sandwich immunoassay for Application of conventional antibody-based assays and further three pathogens: Salmonella spp., Escherichia adaptation in modern biosensor tools show promise for rapid coli O157:H7, and Listeria monocytogenes detection. Most assays are developed for detection of a single • Optimize growth and enrichment of these pathogens target pathogen or toxin. As a result, these methods can be (healthy or stressed) spiked in model food samples in costly when testing for multiple pathogens from a single product a selective enrichment broth for use with microarray. because separate assay methods are required. In addition, a • Evaluate performance of the Pathogen large laboratory space is required to perform separate tests for Enrichment Device (PED). each target pathogen because separate enrichment media and procedures are needed for each pathogen detection method. Project Highlights The development of a single test capable of detecting multiple For simultaneous growth and detection of Salmonella spp., pathogens enriched in a single enrichment media would reduce E. coli O157:H7, and L. monocytogenes, we formulated a costs and yield quick results. This technology would also benefi t selective enrichment broth SEL (Salmonella, E. coli O157:H7, regulatory agencies in the evaluation of food products for key and Listeria) and found it to be suitable for enrichment of all food pathogens. these target pathogens in spiked ready-to-eat deli turkey and salami. In addition, when we evaluated the performance of SEL In the past two decades, several rapid detection methods, for its ability to detect pathogens by immunoassay, we found such as antibody-based, nucleic acid-based, and biochemical- that target pathogens grown in SEL gave a stronger response based methods, were developed. Even though these methods than when they were grown on other media. The global have shortened analysis times, it still takes time for selective protein expression profi les of the three pathogens in SEL were enrichment of samples before conventional rapid detection signifi cantly greater than their respective selective enrichment methods can be employed. Antibody-based methods, such as broths. Proteomic analysis further revealed the presence of a enzyme-immunoassay, require a minimum of 106 CFU/ml for cell unique protein in bacteria when grown on SEL. detection. To achieve that cell level, it is important to use proper enrichment media for detection of foodborne pathogens.

Cell injury or stress encountered during food processing may affect cell numbers. The presence of selective agents in media can delay growth and recovery of stressed or injured cells. Thus, currently used selective enrichment media may not be suitable for use with modern detection methods. Furthermore, methodologies that detect multiple pathogens with a single device are preferred. Therefore, a medium that can support selective enrichment of multiple pathogens is desirable.

“For simultaneous growth and detection of Salmonella spp., E. coli O157:H7, and L. monocytogenes, we formulated a selective enrichment broth SEL (Salmonella, E. coli O157:H7, and Listeria)...” 3 Center for Food Safety Engineering Bhunia

Optical biosensors for food pathogen detection Investigator: Arun Bhunia (Department of Food Science)

Project Rationale Project Highlights The ability of biosensors to detect the presence of pathogens We obtained the antibodies required to detect multiple pathogens or toxins is critical to ensure product safety. Biosensors using a fi ber optic sensor. The antibodies were labeled with employing specifi c antibodies are being widely used and fl uorophor (Alexa-Fluor) and thoroughly characterized according shown to be effective. Our goal was to develop a fi ber optic to their reaction patterns to target pathogens when grown on a sensor using antibodies for detecting foodborne pathogens, multi-pathogen selective enrichment broth, SEL (Salmonella, including Listeria monocytogenes, Escherichia coli O157:H7, E. coli, and Listeria). Initial experimental trials indicate that we and Salmonella Enteritidis. We developed a fi ber optic sensor can detect these three pathogens simultaneously using a fi ber for L. monocytogenes and E. coli O157:H7. We also developed optic sensor. Cross-reactions with heterologous bacteria were a sensitive and specifi c fi ber optic detection assay for S. minimal. Enteritidis in poultry. The assay was compared to time-resolved immunofl uorescence (TRF) for confi rmation. An effi cient multi- pathogen array using a fl ow-through immobilization protocol was also developed for detecting L. monocytogenes, E .coli, and S. Enteritidis. Pilot studies are underway to analyze the binding effi ciencies of an antibody-pathogen complex using selected surface chemistries in order to gain a better understanding of the molecular nature of these interactions. This approach will enable us to increase sensitivity and specifi city of binding on the sensor.

Project Objectives • Develop and evaluate an antibody-coupled fi ber optic biosensor [ANALYTE 2000™] for detecting S. Enteritidis. • Develop an effi cient multi-pathogen array using a fi ber optic biosensor. This requires screening and identifying monoclonal and polyclonal antibodies, developed in our laboratory, for L. monocytogenes, E. coli O157:H7, and S. Enteritidis, and developing effi cient surface chemistry protocols for evaluating and quantifying the binding interactions of an antibody-pathogen complex on the surface of a fi ber optic sensor.

“The ability of biosensors to detect the presence of pathogens or toxins is critical to ensure product safety.” 4 Center for Food Safety Engineering Bhunia, Hirleman and Robinson

Optical forward scattering for bacterial colony diff erentiation and identifi cation Investigators: Arun Bhunia (Department of Food Science), E. Daniel. Hirleman (School of ), J. Paul Robinson (Weldon School of )

Project Rationale • Acquire scatter images of colonies of select The Centers for Disease Control and Prevention (CDC) foodborne bacterial, including pathogens. estimates that 76 million people get sick, more than 300,000 are • Analyze bacterial colonies of different foodborne hospitalized, and 5,000 Americans die each year from foodborne bacteria on non-selective and selective agar media. pathogen infections. Preventing foodborne illnesses remains • Validate the technology by using both inherently a major public health challenge. Listeria monocytogenes, contaminated food samples and samples that have Escherichia coli, and Salmonella are three major foodborne been contaminated with selected pathogens. pathogens of concern in the U.S. L. monocytogenes, along • Analyze cellular composition, cell arrangement, with Salmonella and Toxoplasma, are responsible for more refractive index ,and colony contents using than 75 percent of the foodborne diseases and 1,500 deaths electron microscopy, FT-IR or GC-MS. every year compared to the other known pathogens. There has • Analyze the scatter signal images using “standard feature been an increase in foodborne illnesses, multiple outbreaks, extraction” and “moments of shape analysis” methods. product recalls, and loss of lives as a result of the association Project Highlights with pathogens in processed, ready-to-eat food. Bacterial The most signifi cant accomplishment of fi scal year 2007-2008 contamination in food not only places the public at risk, it is was the design of an automated BARDOT system and related costly to companies due to loss of production time, product algorithm. This system, including hardware and software, was recalls, and liability. redesigned and redeveloped, and a colony counter and locator For detecting and evaluating foods contaminated with L. were constructed to provide colony counts for each plate. To monocytogenes or E. coli, the USDA/FSIS recommends initial achieve that, we added a pair of illumination lights and a CCD enrichment and subsequent plating on a selective agar medium, (cooled coupled device) camera so that automatic colony counts which is often followed by identifi cation procedures. These and the precise location of each colony are visible on a computer procedures are time-consuming, lasting more than fi ve to seven monitor. The automated BARDOT system prototype (which was days. The present industrial demand is to increase the speed manufactured by the local company En’Urga) incorporates the of detection, decrease economical losses, and minimize public colony locator (which locates a colony via a line scanner), the health concerns. Our main objective was to develop a simple forward-scatterometer, and an automated classifi cation package optical light scattering sensory method to reduce the time to into a stand-alone system. identify pathogenic bacteria after plating.

Project Objectives • Design and develop a prototype of a fully automated BActeria Rapid Detection using Optical scattering Technology (BARDOT) system to locate, capture, and classify foodborne pathogenic bacteria.

“The most signifi cant accomplishment ... was the design of an automated BARDOT system and related algorithm.” 5 Center for Food Safety Engineering Cousin and Woloshuk

Immunocapture real-time PCR to detect mycotoxigenic mold spores in grains Investigators: Maribeth A. Cousin (Department of Food Science), Charles P. Woloshuk (Department of Botany and Plant Pathology)

Project Rationale • Determine the specifi city and sensitivity of Currently, there are few commercial rapid methods to detect primer sets and multiplex format. molds and their spores in agricultural commodities, grains, • Optimize the capture of mold spores and release of DNA and foods. In previous research, a protocol was developed to used to detect Fusarium species in foods and grains. identify Fusarium species that produce two major mycotoxins: Project Highlights fumonisins and trichothecenes. This antibody-based method was A procedure was developed and optimized using lyticase developed for Fusarium species to capture antigens of these (Sigma: 5263) to extract DNA from conidia of Fusarium mycotoxin-producers, which were then combined with a real- graminearum and Fusarium verticillioides for use in real-time time PCR assay that was based on species-specifi c and genus- quantitative PCR (qPCR). This was important because the specifi c primers to identify the Fusarium species. The effi ciency methods used to extract DNA from other microorganisms do not of spore capture was limited in the previous research because work for fi lamentous fungi. Lyticase was mixed with buffer and the Fusarium spores were diffi cult to lyse for DNA release. We mercaptoethanol, incubated at 37°C for four or six hours for F. proposed this new research to help resolve that limitation by: graminearum and F. verticillioides, respectively, and shaken in (1) studying physical, enzymatic, and mechanical methods to a bead-beater to physically disrupt the conidia before analyzing break mold spores to release DNA for use in real-time PCR, and with real-time qPCR. By this method, a minimum of 10 conidia (2) incorporating the method developed in objective 1 into the of F. graminearum and 1000 conidia of F. verticillioides could be immunocapture-qPCR method that uses antibodies produced detected. against F. graminearum and F. verticillioides and primers that are specifi c for the Tri6 gene involved in trichothecene biosynthesis and for the Fum1 gene involved in fumonisin biosynthesis. In addition, we proposed to develop a library of PCR primers to other mycotoxigenic genera (Aspergillus that produce afl atoxins and ochratoxin and Penicillium that produce ochratoxin and patulin) for real-time PCR, and to use these primers in multiplex PCR formats to detect all major mycotoxin producers in the same assay. Antibodies to afl atoxin-producing molds and Penicillium species were produced in earlier research.

Project Objectives • Develop primer sets to detect Aspergillus and Penicillium species. • Experiment with different methods to break mold spores of Fusarium species.

“This antibody-based method was developed for Fusarium species to capture antigens of these mycotoxin-producers, which were then combined with a real-time PCR assay...” 6 Center for Food Safety Engineering Irudayaraj, Mauer, DebRoy and Fratamico

Detection of foodborne pathogens via an integrated spectroscopy and biosensor-based approach Investigators: Joseph Irudayaraj (Department of Agricultural and ), Lisa Mauer (Department of Food Science), Chitrita DebRoy (The Pennsylvania State University), Pina Fratamico (USDA)

Project Rationale Project Objectives Zoonotic pathogens such as Salmonella spp., Listeria • Develop and standardize FTIR and raman spectroscopy- monocytogenes, Shiga-toxin producing Escherichia coli based molecular fi ngerprints (spectra) of foodborne (including E. coli O157:H7), and Campylobacter jejuni outbreak strains in conjunction with sampling are recognized as causes of signifi cant and sometimes and regulatory validation in food matrices. lethal foodborne illnesses. Identifi cation of these microbial • Advance infrared equipment, sampling, contaminants is a primary food safety concern in food testing, and validation capabilities for rapid production, processing, and retail environments. Current identifi cation of foodborne pathogens. detection methods for E. coli O157:H7 require enrichment for 18 Project Highlights to 24 hours followed by isolation, prescreening, and confi rmation We developed a FTIR and raman library of spectroscopic with classical biochemical methods or commercially available fi ngerprints for a total of 28 foodborne pathogenic strains. assays based on ELISA, antibody precipitation, or PCR. These Of these, 14 were E. coli O157:H7, and two were outbreak procedures require up to four days to completely identify E. coli strains. We developed a biosensor protocol using gold O157:H7. The infective dose for Salmonella strains varies with nanorods to detect < 10 CFU/ml using a simple ultraviolet-visible the server, food, and person. As few as 1 to 10 cells can cause spectrometer. We fi nalized a procedure to perform component illness, and ranges from 1 to 107 CFU/ml of Salmonella strains analysis to understand further the basis and the origin of the have been reported. FTIR signatures. We constructed and tested the PathoIR chip New technologies for detecting foodborne pathogens that are in the benchtop FTIR. The presence of three signature peaks in rapid, sensitive, and portable with a potential for on-site detection the 850cm-1 to 1100 cm-1 region confi rmed binding of the target are needed to help ensure a safe food supply for consumers. bacteria to the chip surface. We identifi ed a portable system which is twice as sensitive as the current system. This system is The ultimate goal of this research was to develop a portable already remarkably promising. However, we believe that this can miniaturized infrared sensor for specifi c and sensitive detection be further improved. of foodborne pathogens. We proposed to integrate sampling and biosensor modules with Fourier transform infrared/Raman In summary, our key accomplishment this year was the spectroscopy (FTIR/raman) as well as uv-Visible near infrared development of sensitive nanobiosensors that can provide spectroscopy to improve detection sensitivity and specifi city. a detection limit of ~10 CFU/ml using a simple visible–near- The fi rst steps were to constitute the standardization of FTIR infrared spectrometer. The potential exists to further refi ne and raman methodologies with the most appropriate sampling this technology by integrating a pathogen separation element. steps for sensitivity enhancement and biofunctionalization Hence, detection in complex mixtures could be performed steps for specifi city improvement. The second phase focused in one step. This would also facilitate the detection of multiple on extensive validation using food, as well as mock industry pathogens at a very high sensitivity level using a simple samples, and translating the benchtop methodologies to a spectrometer that is affordable and portable. portable mid-infrared device.

“We developed a FTIR and raman library of spectroscopic fi ngerprints for a total of 28 foodborne pathogenic strains.” 7 Center for Food Safety Engineering Irudayaraj

Nanoparticle-based DNA-multiplexed probes for pathogen detection using confocal raman microscopy Investigator: Joseph Irudayaraj (Department of Agricultural and Biological Engineering)

Project Rationale potentially support probing of DNA damage and splicing The overall goal of this research is to develop a probe mechanisms. fabrication and assay synthesis protocol for multiplex DNA detection of food pathogens by surface-enhanced raman Project Objectives scattering (SERS) utilizing non-fl uorescent, label-containing • Investigate the effectiveness and effi ciency nanoparticles as DNA probes. Although research on SERS- of fi ve cheaper non-fl uorescent dyes as labeled DNA examination is very active, it is still in its early raman labels to be used as SERS tags. stages with regard to multiplexing and detecting analytes at • Synthesize SERS-DNA probes for detecting species- low levels. We are capitalizing on the unique spectroscopic specifi c DNA sequences of E. coli O157:H7, signatures (down to ~1 nm resolution) of non-fl uorescing Campylobacter sp., Staphylococcus aureus, Listeria molecules as labels (raman tags) to identify specifi c DNA monocytogenes, and Salmonella sp. as targets. sequences. Because of the distinct fi ngerprint of the labels • Develop a one-pot multiplex detection system using due to SERS, simultaneous detection of multiple DNA the optimized SERS-DNA probe to simultaneously hybridizations without separation is feasible at sub femto molar detect E. coli O157:H7, Campylobacter sp., and (fM) sensitivity. Salmonella sp. in milk and water samples.

Several aspects are unique to this research. We can use Project Highlights multiplex labeling in one system using a range of non- We demonstrated that up to eight non-fl uorescent raman tags fl uorescing labels. A one-pot platform for detection of food can be chosen with distinct signatures for visual multiplexing pathogens at sensitivities not afforded by fl uorescence methods utilizing the SERS spectra. The fabrication step has also been is possible using our approach. Incorporation of a magnetic optimized and detection sensitivity of up to 1 fM is achievable separation step will enable the separation of target sequences for the chosen labels. We demonstrated multiplexing of up to in complex media. Using non-fl uorescent labels [~$10-20/ eight probes for a chosen DNA sequence. We also showed that gm] for multiplexing is many orders cheaper than fl uorescent hybridization of eight different DNA sequences (depicting eight labels [~$10-20/mg]. Furthermore, the choice of SERS labels probes) at one time can be detected. Finally, we developed a is enormous (over 1,000 labels) and extremely sensitive, and strategy to detect DNA sequences in an array (on a glass slide) single-molecule identifi cation has been reported. This implies as well as in a test tube (a one-pot analysis) format. that eventually the detection can be accomplished without the amplifi cation step. In summary, our key accomplishment was the demonstration of an eight-plex nonfl uorescent DNA detection assay using This novel technology, once fully developed, has the potential raman spectroscopy. Steps to standardize the assay for direct to detect multiple analytes in a benchtop setting. Further, SERS detection of target sequences without the PCR simplifi cation probes have the potential to be incorporated into living cells to step is underway. This technique could be utilized as a slide enable real-time monitoring of structural features and electron (lab-on-slide) or tube (lab-on-tube) format for pathogen and transfer processes that occur along the DNA helix. This would disease detection.

“...our key accomplishment was the demonstration of an eight-plex nonfl uorescent DNA detection assay using raman spectroscopy.” 8 Center for Food Safety Engineering Ladisch, Bashir, Bhunia and Robinson

Engineering of biosystems for the detection of Listeria monocytogenes in foods Investigators: Michael R. Ladisch (Department of Agricultural and Biological Engineering), Rashid Bashir (School of Electrical and ), Arun Bhunia (Department of Food Science), J. Paul Robinson (Department of Biomedical Engineering)

Project Rationale vegetables, milk, and meat, and decrease the volume in Pathogenic bacteria cause 90 percent of reported foodborne which the cells are captured by selecting or constructing the appropriate membrane design, then combining with illnesses. One of these pathogens, Listeria monocytogenes, other bioseparation techniques. For validation, use GFP not only causes serious illness, but also can be lethal in infants, engineered cells, as well as non-modifi ed cells, in mixtures people over 60, and immune-compromised individuals. Current of pathogenic and non-pathogenic microorganisms. methods of detecting L. monocytogenes require 15 to 48 hours. • Correlate media composition to changes in growth Many small food processors and producers do not have in- characteristics and metabolism of L. monocytogenes house capabilities to test for food pathogens and must send out cells, and develop media that enhance the response of samples for analysis. This adds up to an additional 24 hours. pathogenic cells to detection methods. A complimentary Overall, two to three days typically elapses between when the objective was to improve low conductivity media food is sampled and when the results are available. The time for the enhanced capture and detection of stressed to result (TTR) is problematic since some foods are consumed cells by antibodies and other bioreceptors. before test results are available. • Combine antibody-based capture and growth detection for the biochip. Design and microfabricate integrated Rapid and affordable technologies to detect L. monocytogenes devices to combine ATP, pH, and/or direct nucleic acid cells directly from food and to distinguish living from dead and antibody-based detection on-chip using multi-channel, cells are needed. This project addresses the fundamental multi-functional designs. The goal was to obtain biochips requirements for developing microchip, bio-based assays that sense multiple parameters simultaneously and that are transportable to the fi eld, useable in a manufacturing improve dielectrophoresis (DEP)-based selective capture of L. monocytogenes and other pathogens in mixtures environment, and capable of rapidly detecting L. of cells and to test sensitivity and selectivity of capture. monocytogenes at the point of use. Our goals were to achieve microscale detection of L. monocytogenes on a real-time or Project Highlights near real-time basis with a TTR of four hours, and to reduce We integrated the multiple functions needed for the the time of culture steps with rapid cell concentration and development and deployment of microfl uidic biochips for recovery based on membrane technology. We are addressing detecting bacterial pathogens. We integrated antibody-based the development and validation of such a microchip system that capture of bacterial cells enhanced by dielectrophoretic combines bioseparations technology—for rapid concentration forces, bacterial culture and electrical detection of bacterial and recovery of microbial cells, and bionanotechnology—to growth, and PCR-based detection of L. monocytogenes—all construct systems capable of interrogating fl uids for pathogens. on chip. A signifi cant accomplishment was the development of label-free electrically amplifi ed PCR products. We showed Project Objectives that impedance can be used to detect the presence of DNA molecules in a solution without any labels directly from • Develop a system for rapid cell concentration and recovery. Improve membrane chemistry and methodology the actual PCR solution. This could lead to user-friendly for handling food samples high in fat and complex miniaturized PCR detection systems that do not need optical molecules presented by blended hotdog, hamburger, labels or optical detectors.

“We showed that impedance can be used to detect the presence of DNA molecules in a solution without any labels directly from the actual PCR solution.” 9 Center for Food Safety Engineering Spotlight on USDA-ARS Scientists

For nearly ten years, scientists at Purdue University and the USDA-ARS Eastern Regional Research Center have established a strong partnership and have collaborated on a number of research and outreach projects. We are proud of the relationship that has continually grown since we joined forces in 1999. For this annual report, we highlight two USDA-ARS scientists that have been instrumental in fostering our overall CFSE effort.

Dr.Dr. AndrewAnd Gehring began his career with the USDA as a T.W. Edminster Postdoctoral Research Associate with ththee ARS’s Eastern Regional Research Center located just outside of Philadelphia. His research focused on dedevelopingv biosensor-based methods for rapid detection of foodborne bacterial pathogens, including immunimmunomagnetico electrochemical methods for Salmonella spp. and E. coli O157:H7. Dr. Gehring later acceptacceptede a permanent position with the ARS as a leather chemist, a seemingly unrelated area of research. HHowever,oweve he eventually began working on rapid dehairing, a food safety initiative during the slaughter of ccattle.attle. PParticipation in this team-based research yielded the generation of a series of publications (one of which wasw selected to be paper of the year in the Journal of the American Leather Chemists Association), an iinternationalnternat patent application, and a gold medal in private sector involvement awarded by the Philadelphia FederaFederall Executive Board. He later returned to his earlier biosensor work on developing rapid methods for post- Dr. Andrew Gehring harvesharvestt food safety analysis where he currently applies multiplexed, antibody-based microarray technology to Research Chemist the raprapid,i high-throughput screening of foods for bacterial pathogens and associated toxins. USDA-ARS ERRC • G Gehring,e A.G., Albin, D.M., Bhunia, A., Reed, S.A., Tu, S., Uknalis, J. Antibody microarray detection of Escherichia coli O157:H7: Quantifi cation, assay limitations, and capture effi ciency. Analytical Chemistry. 2006. v. 78. p. 6601-6607.

• Gehring, A.G., Bailey, D.G., DiMaio, G.L., Crowther, J.C. Improved hide quality and rapid unhairing. Journal of the American Leather Chemists Association. 2002. v. 97. p. 339-348.

Dr. George Paoli earned his Ph.D. from The studying the molecular biology, biochemistry, and physiology of carbon dioxide fi xation in photosynthetic bacteria. He then went to the Air Force Research Laboratory as a National Research Council and Oak Ridge Institute for Scientifi c Education Postdoctoral Fellow to study the microbial biodegradation of nitroaromatic pollutants. After working for two years in the Microbial Biophysics and Residue Chemistry Research Unit at the USDA’s Eastern Regional Research Center as a research associate, Dr. Paoli accepted a permanent position there as a research microbiologist. In his current research, Dr. Paoli employs modern molecular techniques, including antibody phage display, to develop reagents and methods for the detection of microbial pathogens in foods. In particular, Dr. Paoli and his coworkers selected a Listeria monocytogenes-specifi c phage displayed antibody fragment and used this antibody fragment to develop immunomagnetic beads, other immunoreagents, and an optical biosensor for Dr.Dr. GeorGeorgege PaoliPaoli the detection of L. monocytogenes. Dr. Paoli has also applied microbiological, immunological, and PCR-based ResearchResearch methods for the detection and typing of Yersinia pestis, the causative agent of bubonic plague and a possible MicrobioloMicrobiologistgist threat to food security. USDAUSDA-ARS ARS ERRC • Nanduria, V., Bhunia, A.K., Tu, S-I, Paoli, G.C., Brewster, J.D. SPR biosensor for the detection of L. monocytogenes using phage-displayed antibody. Biosensors and Bioelectronics. 2007. v. 23. p. 248-252.

• Paoli, G.C., Kleina, L.G., Brewster, J.D. Development of Listeria monocytogenes specifi c immunomagnetic beads using a single-chain antibody fragment. Foodborne Pathogens and Disease. 2007. v. 4. p. 74-83.

• Paoli, G.C., Bhunia, A.K., Bayles, D.O. Listeria monocytogenes. Fratamico, P.M., Bhunia, A.K., Smith J.L., editors. Caister Academic Press, Wymondham, Norfolk UK. Foodborne Pathogens: Microbiology and Molecular Biology. 2005. p. 295-325.

“We are proud of the relationship that has continually grown since we joined forces in 1999.”

10 Center for Food Safety Engineering Dr. Gehring and Dr. Paoli

Since the inception of the Center for Food Safety Engineering, there has been a strong emphasis on interaction and collaboration with scientists from the UDSA-ARS Eastern Regional Research Center. This collaboration has been fostered through interaction between scientists during annual Purdue-CFSE/ARS-ERRC research planning workshops, annual participation by Purdue-CFSE and ARS-ERRC scientists in the Molecular Detection Symposium during the International Workshop on Rapid Methods and Automation in Microbiology at Kansas State University, and more recently, an invitation for Purdue-CFSE scientists to participate in an ARS-FSIS (Food Safety Inspection Service) research planning workshop, aimed at identifying specifi c FSIS needs.

In addition, ARS scientists have hosted Purdue students and post-doctoral fellows at ERRC who are conducting collaborative research. These collaborative research efforts have been presented at scientifi c meetings and have resulted in several publications in peer-reviewed journals.

Drs. Andrew Gehring and Arun Bhunia have interacted extensively on their mutual interest in the development of a mixed culture enrichment (Salmonella, E. coli O157:H7, Listeria monocytogenes, and Yersinia enterocolitica) in support of their work on multiplexed antibody-based microarray for detection of pathogenic bacteria from food. They have worked both independently and collaboratively on the development of the antibody-based microarray and have co-authored one publication on the subject. As part of her doctoral training, one of Dr. Bhunia’s doctoral students, Kristen Nanchansky, spent a summer at ERRC working with Dr. Gehring developing a chemiluminescent immunoassay for the detection of L. monocytogenes. In addition, Drs. Bhunia and Gehring organized and co-chaired a symposium on Optical Technologies for Industrial, Environmental, and Biological Sensing–Food Safety and Agricultural Monitoring for the International Society for Optical Engineering Photonics East.

Drs. George Paoli and Arun Bhunia share an interest in studying the foodborne pathogen Listeria monocytogenes. Dr. Bhunia has developed and characterized a number of monoclonal antibodies against L. monocytogenes, and Dr. Paoli has applied antibody phage display to select a single-chain antibody specifi c to L. monocytogenes. In addition to co-authoring a chapter on the bacterium in a recently published book on the microbiology and molecular biology of foodborne pathogens, Drs. Paoli and Bhunia collaborated in supervising a post-doctoral study by Dr. Viswaprakash Nanduri on the development of a phage displayed antibody-based surface plasmon resonance optical biosensor for the detection of L. monocytogenes.

International Collaboration: CFSE goes to China

By invitation of Dr. Xianming Shi, the deputy director of the Bor-Luh Chinese Food Safety Center, Purdue scientists Arun Bhunia and Richard Linton joined USDA-ARS scientists (Shu-I Tu, Andrew Gehring, Yiping He, and George Paoli) and Dr. Jim Lindsay (ARS National Program Leader for Food Safety) in China in May 2008 to meet with representatives from Jiao-Tong University in Shanghai. At the meeting an agreement was signed forming a joint U.S.–Sino Food Safety Research Center between ARS and the Chinese Ministry of Science and Technology. We had a very productive meeting and already have plans to work together with USDA-ARS and Jiao-Tong University scientists in the near future. While in Shanghai, the group also attended and presented at the 10th World Congress on Biosensors.

Left: Jim Lindsay, National Program Leader for Food Safety from USDA- ARS, signs an agreement that begins a more formal working relationship with Jaio Tong University in Shanghai, China.

Above: A group photograph with our new colleagues and friends from China.

11 Center for Food Safety Engineering Lu, Bhunia and Cheng

Rapid, quantitative, and reusable immunosensors for bacteria detection on a microfl uidic platform Investigators: Chang Lu (Department of Agricultural and Biological Engineering), Arun Bhunia (Department of Food Science), Zhongyang Cheng (Materials Engineering, Auburn University)

Project Rationale manufacturers and food testing laboratories as well as fi eld- Portable, rapid, and sensitive biosensors for food safety testing activities of governmental agencies. applications enable point-of-care contamination detection and immediate interpretation of the results. In our research Project Objectives project, we proposed to develop an integrated biosensor • Fabricate magnetic nanobars with different sizes and system on a microfl uidic chip for detecting bacteria based geometries and develop protocols for immobilizing on immunoassays. The device will offer a sensitivity of 102 antibodies specifi c to L. monocytogenes on the surface. to 103 bacteria cell detection and an assay time of fewer than The amount of bacterial cells bound to the surface 20 minutes for a single test. Our system will yield quantitative will be characterized under different conditions. data for estimating the number of the target bacterium in a food • Develop an electrophoresis-based immunoassay sample. The microfl uidic system will consist of individual devices coupled with laser-induced fl uorescence on a microfl uidic for cell lysis, lysate purifi cation, and immunoassays. In principle, chip. We will use this tool to quantitatively detect L. the tool will be effective for any bacterium or strain given the monocytogenes based on cell lysate via the interaction availability of a suitable intracellular antigen-antibody pair. In this between alcohol acetaldehyde dehydrogenase (Aad) and its monoclonal antibody (MAb-H7). project, we will demonstrate the concept using an intracellular antigen, alcohol acetaldehyde dehydrogenase (Aad), and its • Demonstrate a prototype-integrated microfl uidic antibody MAb-H7 to detect Listeria monocytogenes. In order to system which incorporates different steps such as manipulation of magnetic nanobars, cell lysis, concentrate L. monocytogenes cells from food samples, we will lysate purifi cation, and immunoassay. fabricate magnetic nanobars with different sizes and geometries and develop protocols for immobilizing antibodies specifi c to L. Project Highlights monocytogenes on the surface. We integrated the concentration, lysis, and competitive immunoassay for detecting L. monocytogenes on a microfl uidic A portable, reusable, and low-cost device would be useful chip. A packed bed of microbeads, with the bead surface coated for point-of-care analysis in the food manufacturing industry. with monoclonal antibody (MAb-H7) that is specifi c to the antigen Conducting bacteria detection tests within food manufacturing Listeria adhesion protein (LAP), was formed in a microfl uidic laboratories would dramatically decrease the turnaround channel to physically trap bacterial cells. Electrical lysis was time for the results and avoid potential contamination and then used to release intracellular materials from the trapped changes in the bacteria during transit. Conventional analytical cells. Fluorescein isothiocyanate (FITC)-labeled LAP was fl owed methods require bulky, expensive equipment that are often through the microbead bed to bind to unreacted antibody sites cost-prohibitive for food manufacturing laboratories. With our and reveal whether LAP was present in the bacterial sample. By lab-on-a-chip approach, sophisticated functions of a biological integrating all these functions onto one simple portable chip, we laboratory can be miniaturized on a microchip, enabling any can produce a biosensor system that is both highly effi cient and minimally equipped laboratory with the ability to perform bacteria inexpensive. detection tests. This technology can signifi cantly benefi t the food industry by enhancing the laboratory-testing capabilities of food

“By integrating all these functions onto one simple portable chip, we can produce a biosensor system that is both highly effi cient and inexpensive.” 12 Center for Food Safety Engineering Morgan and Applegate

A method for capture and detection of Escherichia coli O157:H7 using polymer-immobilized phage Investigators: Mark Morgan (Department of Food Science), Bruce Applegate (Department of Food Science)

Project Rationale Project Highlights Foodborne illnesses in developed nations affect up to 30 During fi scal year 2007, we developed technology supporting percent of the population annually with microbially-associated the production of E. coli O157:H7-specifi c bacteriophage illnesses cost an estimated $7 to $33 billion dollars annually. phiV 10 in a nonpathogenic E. coli host. The phage was More serious is the mortality rate of foodborne and waterborne simply integrated into the genome of a lab strain of E. coli to infectious diseases in developing countries, amounting to form a lysogen, which could subsequently be released in an estimated 2.1 million deaths annually (mostly infants and the programmed expression of recA. This is an extremely children). Despite the increasing incidence of foodborne important breakthrough in phage-based detection in terms of illnesses and contamination, there are currently few if any commercialization. Currently, to produce large quantities of early-detection methodologies which are robust, user-friendly, phage, it must be grown in a specifi c host, which in this case and applicable to widespread use in developing nations. The is E. coli O157:H7. This is problematic for two reasons: fi rst, phage-based technology under development in this project growing 500 liters of E. coli O157:H7 poses a signifi cant risk would circumvent many of the limitations associated with and would require substantially greater monitoring than existing existing technologies by providing a small, inexpensive, fi eld- bacterial-based biotech protocols. Second, if a single E. coli deployable diagnostic platform for detecting minute amounts O157:H7 used in production was found in the commercialized of bacteria in food and, potentially, in clinical diagnostics. assay, it would be catastrophic to the commercialization. The platform is based on the immobilization of a genetically modifi ed bacteriophage specifi c for Escherichia coli O157:H7, which traps and infects the E. coli O157:H7, resulting in the production of a visible red compound which enables visual detection of the pathogen.

Project Objectives • Demonstrate that ultraviolet polymerization is an effective technique to attach phages to polymer surfaces. • Optimize the the effi ciency of the polymerization process. • Modify the phage phiV10 to include luxCDABE and cobA reporters.

“This is an extremely important breakthrough in phage-based detection...” 13 Center for Food Safety Engineering Nivens, Franklin and Corvalan

Continuous monitoring of chemical agents in aqueous media using bioreporter-based sensors Investigators: David Nivens (Department of Food Science), Michael Franklin (Montana State University), Carlos Corvalan (Department of Food Science)

Project Rationale • Construct novel bioreporters with optimal Chemical contamination of our food supply threatens the heath analytical performance for point-of-use and long-term monitoring experiments. of consumers and has become a major concern. As societies become more populated and technologically-advanced, sources • Model the systems to improve all aspects of analytical of pollution and the potential for contamination (inherent, performance and develop application-specifi c biosensors for food and agriculture systems. unintentional, or intentional) are increasing. Inexpensive sensors that have analytical capabilities of detecting harmful Project Highlights chemicals in food would facilitate our nation’s ability to protect Bioreporter-based chemical sensor technology was used to its food supply and minimize health concerns associated with quantify arsenite concentrations (one of the most hazardous contamination. We proposed to develop bioreporter-based forms of arsenic) in liquid food matrices including milk, fruit chemical sensors consisting of genetically programmed cells juices, and bottled water with minimal or no sample preparation. (bioreporters), a disposable cartridge system containing the For example, various amounts of arsenite were spiked into bioreporters, and a detection/communication module for Web- the undiluted apple juice samples (arsenite is a common based and/or networked-based assessment capabilities. With contaminate found in apple orchards). An aliquot of each the successful development of this technology, we anticipate that sample was then exposed to the sensor to generate time- the bioreporter-based chemical sensors will have the analytical dependent linear calibration curves. Results showed that capabilities required to fi ll a critical need in the food industry. apple juice samples with 10 parts per billion (μg/L) arsenite In addition to being potentially inexpensive, these biosensors could be quantifi ed in less than 2 hours. Web- and network- are being developed to detect a hazardous chemical below based software was developed to monitor the responses of the immediately dangerous to health of life (IDHL) limits, minimize bioreporter-based sensors and generate a warning signal when false positives and negatives, have rapid response times, and the analyte concentration exceeded a predetermined alarm be simple to use. We envision that the sensors could be used level. These fi ndings indicate that our technology potentially can with standard food defense practices to further facilitate a safe be used by minimally trained food and agricultural workers to food supply. detect arsenite (and eventually other contaminates) in a liquid food matrix at or below chronic and acute minimal risk levels. Project Objectives • Develop a dual-signaling bioreporter that minimizes false negatives by using bioluminescence and fl uorescence signal for hazardous chemical detection. • Develop a microenvironment that contains programmable cells and nutrients to increase the stability and extend the lifetime of the biosensor.

“...our technology potentially can be used by minimally trained food and agricultural workers to detect arsenite (and eventually other contaminates) in a liquid food matrix...” 14 Center for Food Safety Engineering Park, Leary and Aronson

Field-ready biosensors for high throughput and multiplexed detection of foodborne pathogens Investigators: Kinam Park (Department of Biomedical Engineering), James Leary (Department of Biological Engineering), Arthur Aronson (Department of Biological Sciences)

Project Rationale source, polarizer, a prism, and a detector. A diode laser with a The increased incidence of pathogen-contaminated food places coupled beam expander is our light source. The incident light a new emphasis on the rapid detection and quantifi cation of is t-polarized using a rotating polarizer. A 60°-60°-60° BK7 foodborne pathogens. Therefore, we are developing a surface prism is mounted on a goiniometer, while a BK7 coverslide plasmon resonance (SPR) imaging biosensor for the rapid, label- with a 4x4 array of 50 nm thick gold dots is placed on the top free, and high throughput detection of foodborne pathogens. This surface of the prism. The purpose of the prism is to cause total device integrates an SPR imaging system with a biosensor array internal refl ection that will create the evanescent fi eld necessary immobilized onto a sample surface containing specifi c short for surface plasmon resonance. To capture the refl ectance peptide ligands. A group of short peptides specifi c to certain image, a charge-coupled device (CCD) camera is fi tted with a pathogenic bacteria will be microcontact-printed on a gold chip long working distance 4X microscope objective. The CCD is in linear patterns. This peptide-imprinted gold chip functions as a connected to a laptop computer and PixelScope Professional™ biosensor array for the specifi c detection of unknown foodborne software is used to capture the image from the CCD. pathogens. To determine what fraction of pathogenic bacteria Next we captured the SPR image of a fl uorescent sample and are live or dead and to confi rm the SPR results, we have created validated the SPR image using fl uorescence microscopy. To do a novel hybrid SPR/molecular imaging portable system. this we stained E. coli O157:H7 cultures with the LIVE/DEAD The device would offer a commercial advantage to the food BacLight™ Bacterial Viability Kit. The fl uorescently labeled E. processing industry. It is miniaturized, has fewer components, coli was deposited onto a sensor chip containing a 50 nm thick and is easier to use compared to the current detection systems. layer of gold that had been functionalized with an antibody This biosensor would detect foodborne pathogens present in specifi c to E. coli O157:H7. The bacterial sample was allowed <100 CFU/g of contaminated food within ten minutes. to air dry and SPR images were taken immediately afterward. The slide was then imaged fl uorescently using an inverted Nikon Diaphot fl uorescent microscope fi tted with the appropriate optical Project Objectives fi lters. We fabricated a microfl uidic cell by pouring liquid PDMS • Synthesize and characterize peptides. onto a stereolithographically fabricated photoresist template • Fabricate and characterize a peptide biosensor and then peeling off the PDMS mold. The PDMS fl ow cell was array and microfl uidic fl ow cell. placed on top of the peptide functionalized gold chip and placed • Design and assemble a compact SPR imaging device. on the SPR prism. The capture ligand patterns were generated • Utilize SPR imaging for real-time detection by microcontact printing. of foodborne pathogens. Our most important accomplishment is that we were able to • Optimize the device for high throughput successfully construct the SPR imaging device and acquire both and multiplexed detection. an SPR and fl uorescent molecular image of similar regions of a Project Highlights bacterial sample on the biosensor. We constructed a SPR imaging device using the Kretschmann confi guration. The main components of this device are a light

“... we were able to successfully construct the SPR imaging device and acquire both an SPR and fl uorescent molecular image of similar regions of a bacterial sample on the biosensor.” 15 Center for Food Safety Engineering Stanciu and Andreescu

Portable biosensor for rapid and ultra-sensitive identifi cation of organophoshorous foodborne contaminants Investigators: Lia Stanciu (Department of Materials Engineering), Silvana Andreescu (Clarkson University)

Project Rationale biosensors with enhanced characteristics. We are the fi rst The overall goal of this project was to advance the fi eld of researchers to use this direct binding of enzymes onto Ni-NPs pesticide detection in food by developing ultra-sensitive for this purpose. This accomplishment is important because biosensors based on immobilized acetylcholinesterase (AChE). site-specifi c orientation of enzymes onto electrode surfaces Over the past decades, AChE biosensors have emerged as a has numerous advantages over classical procedures. It is promising technique for food quality control. The development highly sensitive, avoids conformational changes, decreases of these biosensors could complement or replace classical sensor costs (due to a lower enzyme requirement), and entails analytical methods by simplifying or eliminating sample a simple single fabrication step. This method has the potential preparation and making identifi cation of chemical foodborne to become a robust, commercially viable system. contaminants easier and faster, with signifi cant decreases in Alternatively, the procedure combining sol-gel technology with analysis time and cost. screen-printing protocols is also potentially useful for biosensor fabrication. Moreover, the sol-gel method is a versatile and Project Objectives effi cient technique for conserving enzyme activity in organic • Establish the optimum parameters for the immobilization solvents. We expect that this matrix will enable functionality of of AChE and the need and feasibility of using an the enzyme in the presence of organic solvents, if this medium oxidation strategy for phosphorothionates. is necessary to extract the pesticides from the food matrix. • Fabricate and characterize the AChE biosensor by immobilizing the enzyme onto the surface of single-use screen-printing electrodes (SPE). Study enzyme stability and leaching. • Detect pesticides. Obtain calibration plots of the inhibitory degree upon application of various concentrations of pesticides. Determine the detection limit (DL), response time (RT), and linear concentration range (LCR) for selected pesticides. • Assemble and test the biosensor prototype for the analysis of pesticides in food samples. Evaluate the matrix effects and establish whether an extraction step is needed. Project Highlights We have made signifi cant progress toward establishing the optimum parameters for enzyme immobilization using two matrices (sol-gel and Ni-nanoparticles)) that enable preservation of enzymatic activity. The procedure involving attachment via affi nity binding to Ni-nanoparticles is new and highly innovative and can be used to fabricate a new class of

“The development of these biosensors could complement or replace classical analytical methods by simplifying or eliminating sample preparation and making 16 identifi cation of chemical foodborne contaminants easier and faster.” Center for Food Safety Engineering Scientifi c Publications and Presentations

Peer Reviewed Journal Publications (2007-2008) • Kim, Y., Hendrickson, R., Mosier, N. S., Ladisch, M. R., Bals, B., Balan, V., Dale, B.E. Enzyme hydrolysis and ethanol • Bae, E., Banada, P.P., Huff , K., Bhunia, A.K., Robinson, fermentation of liquid hot water and afex pretreated J.P., Hirleman, E.D. Analysis of time-resolved scattering distillers’ grains at high-solids loadings. Bioresource from macroscale bacterial colonies. Journal of Technology. 2008. v. 99(12). p. 5206-5215. Biomedical Optics. 2008. v. 13 (1). p. 014010. • Kim, Y., Mosier, N. S., Hendrickson, R., Ezeji, T., Blaschek, H., Dien, • Banerjee, P., Lenz, D., Robinson, J.P., Rickus, J.L., Bhunia, B., Cotta, M., Dale, B., Ladisch M. R. Composition of corn dry- A.K. A novel and simple cell-based detection system grind ethanol by-products: DDGS, 3 wet cake, and thin stillage. with collagen-encapsulated B-lymphocyte cell line as a Bioresource Technology. 2008. v. 99(12). p. 5165-5176. biosensor for rapid detection of pathogens and toxins. Laboratory Investigation. 2008. v. 88. p. 196-206. • Kim, Y., Mosier, N., Ladisch, M. R. Process simulation of modifi ed dry grind ethanol plant with recycle of • Bao, N., Jagadeesan, B., Bhunia, A.K., Yao, Y., Lu, pretreated and enzymatically hydrolyzed distillers’ grains. C. Quantifi cation of bacterial cells based on Bioresource Technology. 2008. v. 99(12). p. 5177-5192. autofl uorescence on a microfl uidic platform. Journal of Chromatography. 2008. v. 1181. p. 153-158. • Ladisch, M., Dale, B., Tyner, W., Mosier, N.S., Kim, Y., Cotta, M., Dien, B.S., Blaschek, H., Laurenas, E., Shanks, B., Verkade, J., • Bao, N., Lu, C. A microfl uidic device for physical Schell, C., Petersen, G. Cellulose conversion in dry grind ethanol trapping and electrical lysis of bacterial cells. Applied plants. Bioresource Technology. 2008. v. 99(12). p. 5157-5159. Physics Letters. 2008. v. 92. p. 214103. • Ladisch, M. R., Dale, D. Distillers grains: On the • Bao, N., Wang, J., Lu, C. Recent advances in electric pathway to cellulose conversion. Bioresource analysis of cells in microfl uidic systems. Analytical and Technology. 2008. v. 99(12) p. 5155-5156. Bioanalytical Chemistry. 2008. v. 391. p. 933-942. • Lathrop, A.L., Banada, P.P., Bhunia, A.K. Differential • Bhattacharya, S., Salamat, S., Morisette, D., Banada, P., expression of InlB and ActA in Listeria monocytogenes Akin, D., Liu, Y-S., Bhunia, A. K., Ladisch, M., Bashir, R. in selective and nonselective enrichment broths. Journal PCR based-detection in a micro-fabricated platform. of Applied Microbiology. 2008. v. 104. p. 627-639. Lab on a Chip. 2008. v. 8. p. 1130-1136. • Liu, Y-S., Banada, P.P., Bhattacharya, S., Bhunia, A.K., • Bhattacharya, S., Jang, J., Yang, L., Akin, D., Bashir, R. Bashir, R. Electrical characterization of DNA molecules BioMEMS and based approaches for rapid in solution using impedance measurements. Applied detection of biological entities. Journal of Rapid Methods Physics Letters. 2008. v. 92. p. 143902. and Automation in Microbiology. 2007. v. 15. p. 1-32. • Liu, Y-S., Walter, T. M., Chang, W-J., Lim, K-S., Yang, L., • Bhunia, A.K. Biosensors and bio-based methods for the Lee, S-W., Aronson, A., Bashir, R. Electrical detection separation and detection of foodborne pathogens. Advances of germination of model Bacillus Anthracis spores in in Food and Nutrition Research. 2008. v. 54. p. 1-44. microfl uidic biochips. Lab Chip. 2007. v. 7. p. 603-610. • Burgula, Y., Khali, D., Kim, S., Cousin, M.A., Gore, J. P., • Stewart, P.S., Franklin, M.J. Physiological heterogeneity in Reuhs, B.L., Mauer, L.J. Review of mid-IR Fourier- biofi lms. Nature Reviews Microbiology. 2008. v. 6 p.199-210. transform infrared (FT-IR) spectroscopy applications for bacterial detection. Journal of Rapid Methods and • Vermerris, W., Saballos, A., Ejeta, G., Mosier, N. S., Ladisch, Automation in Microbiology. 2007. v. 15. p. 146-175. M. R., Carpita, N. C. Molecular breeding to enhance ethanol production from corn and sorghum stover. Crop Science • Chapple, C., Ladisch, M., Meilan, R. Loosening Society of America. 2007. v. 47(S3). p. S142-S153. Lignin’s grip on biofuel production. Nature Biotechnology. 2007. v. 25 (7). p. 746-748. • Wang, C., Irudayaraj, J. Gold nanorod probes detects multiple pathogens. Small – a • Jedlica, S.S., Little, K.M., Nivens, D.E., Zemlyanov, D., Rickus, Nanotechnology Journal. 2008. (In Press). J.L. Peptide ormosils as cellular substrates. Journal of Materials Chemistry. 2007. v. 17. p. 5058-5067. • Ximenes, E. A., Dien, B. S., Ladisch, M. R., Mosier, N., Cotta, M. A., Li, X. L. Enzyme production by industrially • Kim, H., Bhunia, A.K. SEL, a selective enrichment broth for relevant fungi cultured on feed co-product from corn simultaneous growth of Salmonella enterica, Escherichia dry grind ethanol plants. Applied Biochemistry and coli O157:H7, and Listeria monocytogenes. Applied and Biotechnology. 2007. v. 136-140 (1-12). p. 171-183. Environmental Microbiology. 2008. v. 74 (15). p. 4853-4866. • Yang, L., Banda, P.P., Bhunia, A.K., Bashir, R. Effects of • Kim, G., Morgan, M.T., Ess, D.R., Hahm, B.K., Kothapalli, A., dielectrophoresis on growth, viability, and immuno- Bhunia, A.K. An automated fi ber-optic biosensor based binding reactivity of Listeria monocytogenes. Journal of inhibition assay for the detection of Listeria monocytogenes. Biological Engineering. (2008) v. 2(6). p. Food Science and Biotechnology. 2007. v. 16(3). p. 337-342.

“This year, the CFSE team published 31 peer-reviewed research publications and presented 18 talks at national science meetings.” 17 Center for Food Safety Engineering Scientifi c Publications and Presentations

• Yang, L., Bashir, R. Electrical/electrochemical • Jagadeesan, B., Raizman, E., Nanduri, V., Bannantine, J., Bhunia, impedance for rapid detection of foodborne A.K. Rapid label free sero-diagnosis of Johne’s disease using pathogenic bacteria. Biotechnology Progress, surface plasmon resonance biosensor. American Society for Biotechnology Advances. 2008. v. 26. p. 135-150. Microbiology General Meeting. Boston, MA. June 1-5, 2008. • Yu, C., Irudayaraj, J. Multiplex biosensor using gold nanorods. • Kim, K., Bhunia, A.K. Performance evaluation of a multiplex Analytical Chemistry. 2007. v. 79(2) p. 572-579. selective enrichment broth, SEL, by proteomic analysis and immunoassay. Institute of Food Technologist Annual • Yu, C., Irudayaraj, J. Sensitivity and selectivity Meeting. New Orleans, LA. June 29-July 2, 2008. limits of multiplex nanoSPR biosensor assays. Biophysical Journal. 2007. v. 93(9) p.1-9. • Koo, O. K., Shuaib, S., Ladisch, M. R., Bashir, R., Bhunia, A. K. Targeted capture of pathogenic Listeria using a • Zeng, M. N., Mosier, S., Huang, C-P., Sherman, D. M., Listeria Adhesion Protein (LAP) specifi c mammalian Ladisch, M. R. Microscopic examination of changes cell receptor, Hsp60 for detection of bacteria on of plant cell structure in corn stover due to cellulase biosensor platforms. American Society for Microbiology activity and hot water pretreatment. Biotechnology General Meeting. Boston, MA. June 1-5, 2008. Bioengineerg. 2007. v. 97(2). p. 265-278. • Koo, O. K., Jagadeesan, B., Burkholder, K., Bhunia, A.K. Abstracts for Major Papers/Posters Presented Targeted capture of pathogenic Listeria using a Listeria (2007-2008) Adhesion Protein (LAP) specifi c mammalian cell receptor, Hsp60 for detection of bacteria on biosensor • Banada, P.P., Bernas, T., Robinson, J.P., Bhunia, A. K. platforms. Institute of Food Technologists Annual Proteomic analysis of cytotoxic factors from Bacillus Meeting. Chicago, IL. July 28-August 1, 2007. cereus. American Society for Microbiology General Meeting. Toronto, ON. May 21-25, 2007. • Lenz, A.P., Williamson, K., Franklin M.J. Localized gene expression along vertical transects of Pseudomonas • Bashir, R., Ahmad, I. BioMEMS and bionanotechnology aeruginosa biofi lms. American Society for Microbiology for development of miniaturized instruments. Meeting on Microbial Biofi lms. 2008. Symposium on Center for Analytical Instrumentation Development. West Lafayette, IN. June 18, 2008. • Lenz, A.P., Williamson K., Franklin M.J. Quantifi cation of cell numbers and riboscome content of Pseudomonas aeruginosa • Bashir, R., Bhunia, A., Ladisch, M. Engineering of biosystems biofi lms using laser microdissection and qRT-PCR. General for the detection of Listeria monocytogenes in foods— Meeting of the American Society for Microbiology. 2008. development of a biochip. Manhattan, KS. June 18, 2008. • Liu, Y., Banada, P., Bhattacharya, S., Akin, D., Bhunia, • Bashir, R. Interfacing Silicon and Biology at the A. K., Bashir, R. Electrical characterization of DNA Micro and Nanoscale. NSF USA-EU Workshop on molecules in fl uids using impedance measurements. Bionanotechnology. Ispra, Italy. May, 2008. Biomedical Engineering Society (BMES) Annual • Bashir, R. Interfacing silicon and biology at the micro and Meeting, Los Angeles, CA. September 27-29, 2007. nanoscale. University of Cincinnati, Center • Mishra, K., Burkholder, K. M., Medina-Maldonado, S., Bhunia, Seminar Series. Cincinnati, OH. February 18, 2008. A. K. Cloning, genomic organization and expression of • Bashir, R. BioMEMS and bionanotechnology: Integration of SecA2 Gene in Listeria species. American Society for life sciences and engineering at the micro and nanoscale. Microbiology General Meeting. Boston, MA. June 1-5, 2008. The Knowledge Foundation’s 10th Annual Conference, • Morandage, J. S., Woloshuk, C. P., Cousin, M. A. Release BioDetection Technologies. Atlanta, GA. June 14-15, 2007. of DNA from Fusarium spores for use in real-time PCR. • Bhattacharya, S., Salamat, S., Banada, P., Liu, Y., Institute of Food Technologists. 2008. Abstract p. Morisette, D., Bhunia, A. K., Akin, D., Bashir, R. Integrated • Morandage, J. S., Woloshuk, C. P., Cousin, M. A. detection of microorganisms in a microfl uidic biochip. Enzymatic release of DNA from Fusarium spores Biomedical Engineering Society (BMES) Annual for use in real-time PCR. International Association Meeting. Los Angeles, CA. September 27-29, 2007. for Food Protection. 2008. Abstract p. • Burkholder, K. M., Kim, K.-P., Hahm, B.-K., Mishra, K., Medina- • Nagel, A.C., Schroeder, D.L., Gross, B.D., Co, B., Nivens, D.E. Maldonado, S., Bhunia, A. K. Anaerobic environment Development of an arsenic sensing biosensor as a model for increases surface localization of Listeria Adhesion detection of chemical threat agents in food products. Institute Protein (LAP) and promotes infectivity of Listeria of Food Technologist National Meeting. Chicago, IL. 2007. monocytogenes. American Society for Microbiology General Meeting. Boston, MA. June 1-5, 2008.

“At the Center for Food Safety Engineering, we direct our 18 Center for Food Safety Engineering • Ngamwongsatit, P., Banada, P.P., Bhunia, A.K., Panbangred, • Bhunia, A.K. Microbial Foodborne Pathogens: W. Study on correlation between enterotoxin genes and Mechanisms and Pathogenesis. First Edition, cytotoxicity in Bacillus cereus isolated from patient, food Springer, New York, NY. 2008. 276 p. and soil in Thailand. American Society for Microbiology • Irudayaraj, J., Christoph, R. Nondestructive General Meeting. Boston, MA. June 1-5, 2008. Sensing for Food Quality. IFT Press, Blackwell • Schroeder, D.L., Nagel, A.C., Gross, B.D., Reed, T.S., Ausloos, Publishing Professional. Ames, IA. 2007. D.D., Nivens, D.E. Quantitative detection of arsenic in food • Kim, Y., Hendrickson, R., Mosier, N. S., Ladisch, M. R. using a bioreporter-based chemical sensor. Institute of Food Liquid hot water pretreatment of cellulosic biomass. Technologist National Meeting. New Orleans, LA. 2008. Methods in Molecular Biology. (In press). • Suanthie, Y., Woloshuk, C. P. Multiplex real-time PCR assay • Kizil, R. and Irudayaraj, J. FT-Raman spectroscopy for food to detect and quantify three genera of mycotoxigenic fungi. and biomaterial characterization. Irudayaraj, J., Christoph, Phytopathology. 2007. 2008. v.98. Abstract p. S205. R. editors. IFT Press, Blackwell Publishing Professional, • Sun, L., Irudayaraj, J. Confocal raman based nanoarray Ames, IA. Nondestructive Sensing for Food Quality. 2007. platform for multiplex detection using non-fl uorescent • Kizil, R., Irudayaraj, J. FT-Raman spectroscopy for food and tags. The 12th Annual Meeting of the Institute of biomaterial characterization. Elsevier Publications Ltd. In Biological Engineering. March 29-April 1. Modern Techniques for Food Authentication. (In Press) • Walker, S., Heinemann, P., Catchmark, J., Debroy., • Ray, B., Bhunia, A.K. Fundamental Food Microbiology. C., Irudayaraj, J. Detection of Escheria Coli using a 4th Edition. CRC Press, Taylor and Francis novel scanning imaging surface Plasmon resonance group, Boca Raton, FL. 2008. 491 p. biosensor. The 12th Annual Meeting of the Institute of Biological Engineering March 29-April 1, 2007. • Schwietzke, S., Kim, Y., Ximenes, E., Mosier, N., Ladisch, M. Ethanol production from maize. Brian Larkins, editor. • Woloshuk, C.P., Suanthie, Y. Real time PCR assay applications Biomass for Maize Biotechnology. (In press). for distillers grain. Midwest Section Meeting of the Association of Analytical Communities International. 2008. Abstract p. 18. • Wang, H.Y., Banada, P.P., Bhunia, A.K., Lu, C. Rapid electrical lysis of bacterial cells in a microfl uidic device. Floriano, Theses/Dissertations (2007-2008) P.N., editor. Humana Press, Totowa, NJ. Methods in Molecular Biology vol. 385: Microchip-based Assay • Banerjee, P. Mammalian cell based biosensor for rapid Systems: Methods and Applications. 2007. p. 23-35. screening of pathogenic bacteria and toxins. Ph.D. Dissertation. 2008. Purdue University. 191 p. • Yu, C., Irudayaraj, J. Sensitivity and selectivity limits of multiplex nanoSPR biosensor assays. Nagarajan, R. • Huff , K. The light scatterometer BARDOT as a noninvasive editor. American Chemical Society Books, American sensor for the identifi cation of common foodborne Chemical Society. Nanoparticles: Synthesis, Passivation, bacteria. M.S. Thesis. 2008. Purdue University. 172 p. Stabilization, and Functionalization. 2007. • Liu, Y.-S. Impedance spectroscopy based micro-scale • Zeng, M., Ladisch, M. R. Breaking barriers to cellulosic biosensing. Ph.D. Thesis. 2008. Purdue University. ethanol: understanding interactions between • Shin, J. Biobattery. Ph.D. Thesis. 2008. Purdue University. modifi cations of plant cells, enzymes and pretreatments. Plant Biotechnology Reviews. (In press). • Wang, H.Y. Microfl uidic electroporation and cell arrays. Ph.D. Dissertation. 2007. Purdue University. 166 p. Patents Granted (2007-2008) Books and Book Chapters (2007-2008) • Hirleman, E.D., Guo, S., Bae, E., Bhunia, A.K. System and method for rapid detection and characterization of bacterial colonies • Banada, P.P., Bhunia, A.K. Antibodies and immunoassays using forward light scattering. U.S. Patent 64142.00. for detection of bacterial pathogens. Zourob, M., Turner, P.F., editors. Cambridge University, • Multiplex biosensor using gold nanostructures. 2007. Manchester, UK. Section II. Biorecognition. New U.S. Provisional Patent Application 64803.P1.US. Technologies for Bacterial Pathogen Detection. • Purdue Research Foundation. Identity profi ling • Bao, N., Lu, C. Microfl uidics-based lysis of bacteria of cell surface markers. 2008. U.S. Provisional and spores for detection and analysis. Zourob, M., Patent Application 64892.P2.US. Elwary, S., Turner, A., editors. Springer, New York, NY. • Gomez, R., Bashir, R., Bhunia, A. K., Ladisch, M., Principles of Bacterial Detection: Biosensors, Recognition Robinson, J. P. Biosensor and Related Method. Receptors and Microsystems. 2008. p. 783-796. 2007. U.S. Patent 7,306,924 US. efforts toward detecting problems and protecting consumers.” 19 Center for Food Safety Engineering Center Staff

Dr. Arun K. Bhunia Co-PIs PI Amornrat Aroonnual • 765.496.3826 • [email protected] 765.494.5443 Euiwon Bae • 765.494.4762 • [email protected] [email protected] Andrew Gehring • 215.233.6491 • [email protected] E. Daniel Hirleman • 765.494.5688 • [email protected] Seung Ohk • 765.496.7356 • [email protected] Valery Patsekin • 765.494.0757 • [email protected] Bartek Rajwa • 765.494.0757 • rajwa@fl owcyt.cyto.purdue.edu J. Paul Robinson • 765.494.6449 • jpr@fl ocyt.cyto.purdue.edu Shu-I-Tu • 215.233.6466 • [email protected] Staff / Graduate Students Nan Bai • [email protected] Amanda Bettasso • [email protected] Hyochin Kim • 765.496.7354 • [email protected] Dr. Maribeth A. Cousin Co-PI PI Charles P. Woloshuk • 765.494.3450 • [email protected] 765.494.8287 [email protected] Staff / Graduate Students Yenny Suanthie • [email protected] Janaka Morandage • [email protected]

Dr. Joseph Irudayaraj Co-PIsCo-PIs PI ChitritaChitrit DebRoy • [email protected] 765.494.0388 PIna Fratamico F • [email protected] [email protected] Lisa Mauer • 765.494.9111 • [email protected] Staff / Graduate Students Deepali Herlekar • [email protected] Sandeep Ravindran • [email protected] Chungang Wang • 765.494.0388 • [email protected]

Dr. Chang Lu Co-PIs PI Ning Bao • [email protected] 765.494.1188 Arun Bhunia • 765.494.5443 • [email protected] [email protected] Zhongyang Cheng • [email protected] Staff / Graduate Students Hsiang-Yu Wang • [email protected] Balamurugan Jagadessan • [email protected] Peixuan Wu • [email protected]

Dr. Mark Morgan Co-PI PI Bruce Applegate • 765.496.7920 • [email protected] 765.494.1180 [email protected] Staff Lynda Perry • 765.494.7698 • [email protected]

20 Center for Food Safety Engineering Dr. Michael Ladisch Co-PIs PI Amornrat Aroonnual • 765.496.3826 • [email protected] 765.494.7022 Rashid Bashir • [email protected] [email protected] Arun Bhunia • 765.494.5443 • [email protected] Youngmi Kim • [email protected] Xingya Liu • 765.494.-7052 • [email protected] Krishna Mishra • 765.494.6236 • [email protected] Nathan Mosier • 765.494.7025 • [email protected] J. Paul Robinson • 765.494.6449 • [email protected] Andres Rodriguez • [email protected] Eduardo Ximenes • [email protected] Miroslav Sedlak • 765.494.3699 • [email protected] Staff / Graduate Students Kristin Burkholder • 765.496.7354 • [email protected] Bala Jagadeesan • 765.496.7356 • [email protected] Ok Kyung Koo • 765.496.7354 • [email protected] Yi-Shao Liu • [email protected] Jaeho Shin • [email protected] David Sung • [email protected] Shuaib Salamat • [email protected] Stefan Schwietzke • [email protected] Angela Valadez • 765.496.3824 • [email protected]

Dr. David E. Nivens Co-PIs PI Carlos Corvalan • 765.494.8262 • [email protected] 765.494.0460 Michael Franklin • 406.994.5658 • [email protected] [email protected] Staff / Graduate Students Ben Gross • 765.494.6960 • [email protected] Claudia Ionita • 765.496.7354 • [email protected] Ailyn Lenz • [email protected] Aaron Nagel • 765.496.3832 • [email protected]

Dr. Kinam Park Co-PIs PI James F. Leary • 765.494.7280 • jfl [email protected] 765.494.7759 Arthur I. Aronson • [email protected] [email protected] Ghanashyam Acharya • [email protected] Jong-Ho Kim • [email protected] Staff / Graduate Students Meggie Grafton • 765.494.2955 • [email protected] Michael Zordan • [email protected]

Dr. Lia Stanciu Co-PI PI Dr. Silvana Andreescu • 315.268.2394 • [email protected] 765.496.3552 [email protected] Staff / Graduate Students Mallikarjunarao Ganesana • 315.368.3806 • [email protected] Brian Frederick • 315.368.2348 • [email protected]

21 Center for Food Safety Engineering It is the policy of the Purdue University Center for Food Safety Engineering, that all persons shall have equal opportunity and access to the programs and facilities without regard to race, color, sex, religion, national origin, age, marital status, parental status, sexual orientation or disability.

Purdue University is an Affi rmative Action employer.

The Center for Food Safety Engineering Non-profi t Organization Purdue University U. Postage Food Science Building PAID 745 Agriculture Mall Drive Purdue University West Lafayette, IN 47909