Annual Report - Center for Food Safety Engineering - Purdue ...

2007-2008 Research Report2 Welcome from the Director• Message from Richard Linton, Center Director2 Message from USDA• Message from our Partnership with USDA-ARS3 Multipathogen screening using immunomicroarray• Arun Bhunia4 Optical biosensors for food pathogen detection• Arun Bhunia5 Optical forward scattering for bacterial colony differentiation and identifi cation• Arun Bhunia, E. Daniel. Hirleman, J. Paul Robinson6 Immunocapture real-time PCR to detect mycotoxigenic mold spores in grains• Maribeth A. Cousin, Charles P. Woloshuk7 Detection of foodborne pathogens via an integratedspectroscopy and biosensor-based approach• Joseph Irudayaraj, Lisa Mauer, Chitrita DebRoy, Pina Fratamico8 Nanoparticle-based DNA-multiplexed probes for pathogendetection using confocal raman microscopy• Joseph Irudayaraj9 Engineering of biosystems for the detection of Listeria monocytogenes in foods• Michael R. Ladisch, Rashid Bashir, Arun Bhunia, J. Paul Robinson10 Spotlight on USDA-ARS Scientists12 Rapid, quantitative, and reusable immunosensors forbacteria detection on a microfl uidic platform• Chang Lu, Arun Bhunia, Zhongyang Cheng13 A method for capture and detection of Escherichia coliO157:H7 using polymer-immobilized phage• Mark Morgan, Bruce Applegate14 Continuous monitoring of chemical agents in aqueousmedia using bioreporter-based sensors• David Nivens, Michael Franklin, Carlos Corvalan15 Field-ready biosensors for high throughput and multiplexeddetection of foodborne pathogens• Kinam Park, James Leary, Arthur Aronson16 Portable biosensor for rapid and ultra-sensitive identifi cationof organophoshorous foodborne contaminants• Lia Stanciu, Silvana Andreescu17 Scientifi c Publications and Presentations20 Center StaffVisit us @ www.cfse.purdue.edu

Welcome from the DirectorThe 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-AgriculturalResearch Service (USDA-ARS) Eastern Regional Research Center continues to create signifi cantresearch and outreach impacts. This year, the CFSE team published 31 peer-reviewed researchpublications and presented 18 talks at national science meetings. CFSE scientists also havebeen forming partnerships with international countries. Scientifi c presentations and collaborationseekingvisits were made this year to Brazil, China, Hong Kong, and Scotland. Our trip to Jiao-TongUniversity in Shanghai, China was especially important in fostering a new international collaborationbetween USDA-ARS and Jiao-Tong University.Dr. Richard H. LintonDirector of the Center forFood Safety Engineering765.494.64816481linton@purdue.eduThis year, our research program underwent a USDA-ARS Offi ce of Scientifi c Quality Review(OSQR), and the future direction of our research program was identifi ed. A research priority forthe next few years is development of technological platforms that improve microbial and chemicalhazard detection. These new technologies will advance detection of bacterial pathogens includingListeria monocytogenes, Escherichia coli O157:H7, Campylobacter spp., and Salmonella spp. andof chemical hazards that may present food safety and food defense concerns.Our detection-based technologies will build upon prior success with optical biosensors, cellbasedbiosensors, bio-chips (lab-on-a-chip), microarrays, infrared spectroscopy (includingFourier transform infrared FTIR), enzyme linked immunosorbant assays, polymerase chainreactions, impedance-based microbiology, scanning microscopy, confocal raman microscopy,bioluminescence, DNA/RNA probes, and bioreporter-based chemical sensors. The exciting BActeriaRapid Detection using Optical scattering Technology (BARDOT) system is being evaluated byindustry and regulatory agencies, and we hope that it will be available for wide-scale use sometimenext 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 learningmore about CFSE, please visit our Web site at www.cfse.purdue.edu or contact me directly.Message from USDADr. Shu-I TuSupervisory ResearchChemist USDA-ARS,Eastern RegionalResearch Center215.233.6466shui.tu@ars.usda.govAs the collaboration between the CFSE at Purdue University and the USDA-ARS EasternRegional 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 hassuccessfully completed the USDA-ARS OSQR process with outstanding ratings. This resultsignals that the ARS-Purdue project is now considered to be an integral part of USDA-ARSresearch efforts. Together, we have received increased recognition as an important contributorto the technological advancement of pathogen detection in food—evidenced by the fact that theteam received invitations from the International Workshop on Rapid Methods and Automation inMicrobiology to conduct half-day symposiums on molecular methodologies in August 2006, June2007, and June 2008. The team received extraordinary praise from Dr. Daniel Y. C. Fung, Professorof Food Microbiology, Kansas State University, for invaluable contributions to the workshop, andhe extended an invitation for our continuing involvement in 2009. In May 2008, our team went toShanghai, China to attend the fi rst annual meeting of the Joint United States-China Food SafetyCenter, an international collaboration established between the ARS and Jiao-Tong University inShanghai. This collaboration is part of the cooperative research activities between the USDA andthe Ministry of Science and Technology, China (MOST). With this collaboration in place, I believethat our ARS-Purdue team will become an important international research enterprise in the nearfuture.2Center for Food Safety Engineering

BhuniaOptical biosensors for food pathogen detectionInvestigator: Arun Bhunia (Department of Food Science)Project RationaleThe ability of biosensors to detect the presence of pathogensor toxins is critical to ensure product safety. Biosensorsemploying specifi c antibodies are being widely used andshown to be effective. Our goal was to develop a fi ber opticsensor using antibodies for detecting foodborne pathogens,including Listeria monocytogenes, Escherichia coli O157:H7,and Salmonella Enteritidis. We developed a fi ber optic sensorfor L. monocytogenes and E. coli O157:H7. We also developeda sensitive and specifi c fi ber optic detection assay for S.Enteritidis in poultry. The assay was compared to time-resolvedimmunofl uorescence (TRF) for confi rmation. An effi cient multipathogenarray using a fl ow-through immobilization protocol wasalso developed for detecting L. monocytogenes, E .coli, and S.Enteritidis. Pilot studies are underway to analyze the bindingeffi ciencies of an antibody-pathogen complex using selectedsurface chemistries in order to gain a better understanding of themolecular nature of these interactions. This approach will enableus to increase sensitivity and specifi city of binding on the sensor.Project HighlightsWe obtained the antibodies required to detect multiple pathogensusing a fi ber optic sensor. The antibodies were labeled withfl uorophor (Alexa-Fluor) and thoroughly characterized accordingto their reaction patterns to target pathogens when grown on amulti-pathogen selective enrichment broth, SEL (Salmonella,E. coli, and Listeria). Initial experimental trials indicate that wecan detect these three pathogens simultaneously using a fi beroptic sensor. Cross-reactions with heterologous bacteria wereminimal.Project Objectives• Develop and evaluate an antibody-coupled fi ber opticbiosensor [ANALYTE 2000] for detecting S. Enteritidis.• Develop an effi cient multi-pathogen array using a fi beroptic biosensor. This requires screening and identifyingmonoclonal and polyclonal antibodies, developed inour laboratory, for L. monocytogenes, E. coli O157:H7,and S. Enteritidis, and developing effi cient surfacechemistry protocols for evaluating and quantifyingthe binding interactions of an antibody-pathogencomplex on the surface of a fi ber optic sensor.4Center for Food Safety Engineering“The ability of biosensors to detect the presence of pathogensor toxins is critical to ensure product safety.”

Bhunia, Hirleman and RobinsonOptical forward scattering for bacterial colonydifferentiation and identificationInvestigators: Arun Bhunia (Department of Food Science), E. Daniel. Hirleman (School of MechanicalEngineering), J. Paul Robinson (Weldon School of Biomedical Engineering)Project RationaleThe Centers for Disease Control and Prevention (CDC)estimates that 76 million people get sick, more than 300,000 arehospitalized, and 5,000 Americans die each year from foodbornepathogen infections. Preventing foodborne illnesses remainsa major public health challenge. Listeria monocytogenes,Escherichia coli, and Salmonella are three major foodbornepathogens of concern in the U.S. L. monocytogenes, alongwith Salmonella and Toxoplasma, are responsible for morethan 75 percent of the foodborne diseases and 1,500 deathsevery year compared to the other known pathogens. There hasbeen an increase in foodborne illnesses, multiple outbreaks,product recalls, and loss of lives as a result of the associationwith pathogens in processed, ready-to-eat food. Bacterialcontamination in food not only places the public at risk, it iscostly to companies due to loss of production time, productrecalls, and liability.For detecting and evaluating foods contaminated with L.monocytogenes or E. coli, the USDA/FSIS recommends initialenrichment and subsequent plating on a selective agar medium,which is often followed by identifi cation procedures. Theseprocedures are time-consuming, lasting more than fi ve to sevendays. The present industrial demand is to increase the speedof detection, decrease economical losses, and minimize publichealth concerns. Our main objective was to develop a simpleoptical light scattering sensory method to reduce the time toidentify pathogenic bacteria after plating.• Acquire scatter images of colonies of selectfoodborne bacterial, including pathogens.• Analyze bacterial colonies of different foodbornebacteria on non-selective and selective agar media.• Validate the technology by using both inherentlycontaminated food samples and samples that havebeen contaminated with selected pathogens.• Analyze cellular composition, cell arrangement,refractive index ,and colony contents usingelectron microscopy, FT-IR or GC-MS.• Analyze the scatter signal images using “standard featureextraction” and “moments of shape analysis” methods.Project HighlightsThe most signifi cant accomplishment of fi scal year 2007-2008was the design of an automated BARDOT system and relatedalgorithm. This system, including hardware and software, wasredesigned and redeveloped, and a colony counter and locatorwere constructed to provide colony counts for each plate. Toachieve that, we added a pair of illumination lights and a CCD(cooled coupled device) camera so that automatic colony countsand the precise location of each colony are visible on a computermonitor. The automated BARDOT system prototype (which wasmanufactured by the local company En’Urga) incorporates thecolony locator (which locates a colony via a line scanner), theforward-scatterometer, and an automated classifi cation packageinto a stand-alone system.Project Objectives• Design and develop a prototype of a fully automatedBActeria Rapid Detection using Optical scatteringTechnology (BARDOT) system to locate, capture,and classify foodborne pathogenic bacteria.“The most signifi cant accomplishment ... was the design of anautomated BARDOT system and related algorithm.”5Center for Food Safety Engineering

Cousin and WoloshukImmunocapture real-time PCR to detectmycotoxigenic mold spores in grainsInvestigators: Maribeth A. Cousin (Department of Food Science), Charles P. Woloshuk (Department of Botany and Plant Pathology)Project RationaleCurrently, there are few commercial rapid methods to detectmolds and their spores in agricultural commodities, grains,and foods. In previous research, a protocol was developed toidentify Fusarium species that produce two major mycotoxins:fumonisins and trichothecenes. This antibody-based method wasdeveloped for Fusarium species to capture antigens of thesemycotoxin-producers, which were then combined with a realtimePCR assay that was based on species-specifi c and genusspecific primers to identify the Fusarium species. The effi ciencyof spore capture was limited in the previous research becausethe Fusarium spores were diffi cult to lyse for DNA release. Weproposed this new research to help resolve that limitation by:(1) studying physical, enzymatic, and mechanical methods tobreak mold spores to release DNA for use in real-time PCR, and(2) incorporating the method developed in objective 1 into theimmunocapture-qPCR method that uses antibodies producedagainst F. graminearum and F. verticillioides and primers that arespecifi c for the Tri6 gene involved in trichothecene biosynthesisand for the Fum1 gene involved in fumonisin biosynthesis. Inaddition, we proposed to develop a library of PCR primers toother mycotoxigenic genera (Aspergillus that produce afl atoxinsand ochratoxin and Penicillium that produce ochratoxin andpatulin) for real-time PCR, and to use these primers in multiplexPCR formats to detect all major mycotoxin producers in thesame assay. Antibodies to afl atoxin-producing molds andPenicillium species were produced in earlier research.• Determine the specifi city and sensitivity ofprimer sets and multiplex format.• Optimize the capture of mold spores and release of DNAused to detect Fusarium species in foods and grains.Project HighlightsA procedure was developed and optimized using lyticase(Sigma: 5263) to extract DNA from conidia of Fusariumgraminearum and Fusarium verticillioides for use in real-timequantitative PCR (qPCR). This was important because themethods used to extract DNA from other microorganisms do notwork for fi lamentous fungi. Lyticase was mixed with buffer andmercaptoethanol, incubated at 37°C for four or six hours for F.graminearum and F. verticillioides, respectively, and shaken ina bead-beater to physically disrupt the conidia before analyzingwith real-time qPCR. By this method, a minimum of 10 conidiaof F. graminearum and 1000 conidia of F. verticillioides could bedetected.Project Objectives• Develop primer sets to detect Aspergillusand Penicillium species.• Experiment with different methods to breakmold spores of Fusarium species.6Center for Food Safety Engineering“This antibody-based method was developed for Fusarium species to capture antigens ofthese mycotoxin-producers, which were then combined with a real-time PCR assay...”

Irudayaraj, Mauer, DebRoy and FratamicoDetection of foodborne pathogens via an integratedspectroscopy and biosensor-based approachInvestigators: Joseph Irudayaraj (Department of Agricultural and Biological Engineering), Lisa Mauer (Department of FoodScience), Chitrita DebRoy (The Pennsylvania State University), Pina Fratamico (USDA)Project RationaleZoonotic pathogens such as Salmonella spp., Listeriamonocytogenes, Shiga-toxin producing Escherichia coli(including E. coli O157:H7), and Campylobacter jejuniare recognized as causes of signifi cant and sometimeslethal foodborne illnesses. Identifi cation of these microbialcontaminants is a primary food safety concern in foodproduction, processing, and retail environments. Currentdetection methods for E. coli O157:H7 require enrichment for 18to 24 hours followed by isolation, prescreening, and confi rmationwith classical biochemical methods or commercially availableassays based on ELISA, antibody precipitation, or PCR. Theseprocedures require up to four days to completely identify E. coliO157:H7. The infective dose for Salmonella strains varies withthe server, food, and person. As few as 1 to 10 cells can causeillness, and ranges from 1 to 10 7 CFU/ml of Salmonella strainshave been reported.New technologies for detecting foodborne pathogens that arerapid, sensitive, and portable with a potential for on-site detectionare needed to help ensure a safe food supply for consumers.The ultimate goal of this research was to develop a portableminiaturized infrared sensor for specifi c and sensitive detectionof foodborne pathogens. We proposed to integrate samplingand biosensor modules with Fourier transform infrared/Ramanspectroscopy (FTIR/raman) as well as uv-Visible near infraredspectroscopy to improve detection sensitivity and specifi city.The fi rst steps were to constitute the standardization of FTIRand raman methodologies with the most appropriate samplingsteps for sensitivity enhancement and biofunctionalizationsteps for specifi city improvement. The second phase focusedon extensive validation using food, as well as mock industrysamples, and translating the benchtop methodologies to aportable mid-infrared device.Project Objectives• Develop and standardize FTIR and raman spectroscopybasedmolecular fi ngerprints (spectra) of foodborneoutbreak strains in conjunction with samplingand regulatory validation in food matrices.• Advance infrared equipment, sampling,testing, and validation capabilities for rapididentifi cation of foodborne pathogens.Project HighlightsWe developed a FTIR and raman library of spectroscopicfi ngerprints for a total of 28 foodborne pathogenic strains.Of these, 14 were E. coli O157:H7, and two were outbreakstrains. We developed a biosensor protocol using goldnanorods to detect < 10 CFU/ml using a simple ultraviolet-visiblespectrometer. We fi nalized a procedure to perform componentanalysis to understand further the basis and the origin of theFTIR signatures. We constructed and tested the PathoIR chipin the benchtop FTIR. The presence of three signature peaks inthe 850cm -1 to 1100 cm -1 region confi rmed binding of the targetbacteria to the chip surface. We identifi ed a portable systemwhich is twice as sensitive as the current system. This system isalready remarkably promising. However, we believe that this canbe further improved.In summary, our key accomplishment this year was thedevelopment of sensitive nanobiosensors that can providea detection limit of ~10 CFU/ml using a simple visible–nearinfraredspectrometer. The potential exists to further refi nethis technology by integrating a pathogen separation element.Hence, detection in complex mixtures could be performedin one step. This would also facilitate the detection of multiplepathogens at a very high sensitivity level using a simplespectrometer that is affordable and portable.“We developed a FTIR and raman library of spectroscopicfi ngerprints for a total of 28 foodborne pathogenic strains.”7Center for Food Safety Engineering

IrudayarajNanoparticle-based DNA-multiplexed probes for pathogendetection using confocal raman microscopyInvestigator: Joseph Irudayaraj (Department of Agricultural and Biological Engineering)Project RationaleThe overall goal of this research is to develop a probefabrication and assay synthesis protocol for multiplex DNAdetection of food pathogens by surface-enhanced ramanscattering (SERS) utilizing non-fl uorescent, label-containingnanoparticles as DNA probes. Although research on SERSlabeledDNA examination is very active, it is still in its earlystages with regard to multiplexing and detecting analytes atlow levels. We are capitalizing on the unique spectroscopicsignatures (down to ~1 nm resolution) of non-fl uorescingmolecules as labels (raman tags) to identify specific DNAsequences. Because of the distinct fi ngerprint of the labelsdue to SERS, simultaneous detection of multiple DNAhybridizations without separation is feasible at sub femto molar(fM) sensitivity.Several aspects are unique to this research. We can usemultiplex labeling in one system using a range of nonfluorescing labels. A one-pot platform for detection of foodpathogens at sensitivities not afforded by fl uorescence methodsis possible using our approach. Incorporation of a magneticseparation step will enable the separation of target sequencesin complex media. Using non-fl uorescent labels [~$10-20/gm] for multiplexing is many orders cheaper than fl uorescentlabels [~$10-20/mg]. Furthermore, the choice of SERS labelsis enormous (over 1,000 labels) and extremely sensitive, andsingle-molecule identifi cation has been reported. This impliesthat eventually the detection can be accomplished without theamplifi cation step.This novel technology, once fully developed, has the potentialto detect multiple analytes in a benchtop setting. Further, SERSprobes have the potential to be incorporated into living cells toenable real-time monitoring of structural features and electrontransfer processes that occur along the DNA helix. This wouldpotentially support probing of DNA damage and splicingmechanisms.Project Objectives• Investigate the effectiveness and effi ciencyof fi ve cheaper non-fl uorescent dyes asraman labels to be used as SERS tags.• Synthesize SERS-DNA probes for detecting speciesspecific DNA sequences of E. coli O157:H7,Campylobacter sp., Staphylococcus aureus, Listeriamonocytogenes, and Salmonella sp. as targets.• Develop a one-pot multiplex detection system usingthe optimized SERS-DNA probe to simultaneouslydetect E. coli O157:H7, Campylobacter sp., andSalmonella sp. in milk and water samples.Project HighlightsWe demonstrated that up to eight non-fl uorescent raman tagscan be chosen with distinct signatures for visual multiplexingutilizing the SERS spectra. The fabrication step has also beenoptimized and detection sensitivity of up to 1 fM is achievablefor the chosen labels. We demonstrated multiplexing of up toeight probes for a chosen DNA sequence. We also showed thathybridization of eight different DNA sequences (depicting eightprobes) at one time can be detected. Finally, we developed astrategy to detect DNA sequences in an array (on a glass slide)as well as in a test tube (a one-pot analysis) format.In summary, our key accomplishment was the demonstrationof an eight-plex nonfl uorescent DNA detection assay usingraman spectroscopy. Steps to standardize the assay for directdetection of target sequences without the PCR simplifi cationstep is underway. This technique could be utilized as a slide(lab-on-slide) or tube (lab-on-tube) format for pathogen anddisease detection.8Center for Food Safety Engineering“...our key accomplishment was the demonstration of an eight-plexnonfl uorescent DNA detection assay using raman spectroscopy.”

Ladisch, Bashir, Bhunia and RobinsonEngineering of biosystems for the detectionof Listeria monocytogenes in foodsInvestigators: Michael R. Ladisch (Department of Agricultural and Biological Engineering), Rashid Bashir (School of Electrical and ComputerEngineering), Arun Bhunia (Department of Food Science), J. Paul Robinson (Department of Biomedical Engineering)Project RationalePathogenic bacteria cause 90 percent of reported foodborneillnesses. One of these pathogens, Listeria monocytogenes,not only causes serious illness, but also can be lethal in infants,people over 60, and immune-compromised individuals. Currentmethods of detecting L. monocytogenes require 15 to 48 hours.Many small food processors and producers do not have inhousecapabilities to test for food pathogens and must send outsamples for analysis. This adds up to an additional 24 hours.Overall, two to three days typically elapses between when thefood is sampled and when the results are available. The timeto result (TTR) is problematic since some foods are consumedbefore test results are available.Rapid and affordable technologies to detect L. monocytogenescells directly from food and to distinguish living from deadcells are needed. This project addresses the fundamentalrequirements for developing microchip, bio-based assaysthat are transportable to the fi eld, useable in a manufacturingenvironment, and capable of rapidly detecting L.monocytogenes at the point of use. Our goals were to achievemicroscale detection of L. monocytogenes on a real-time ornear real-time basis with a TTR of four hours, and to reducethe time of culture steps with rapid cell concentration andrecovery based on membrane technology. We are addressingthe development and validation of such a microchip system thatcombines bioseparations technology—for rapid concentrationand recovery of microbial cells, and bionanotechnology—toconstruct systems capable of interrogating fl uids for pathogens.Project Objectives• Develop a system for rapid cell concentration andrecovery. Improve membrane chemistry and methodologyfor handling food samples high in fat and complexmolecules presented by blended hotdog, hamburger,vegetables, milk, and meat, and decrease the volume inwhich the cells are captured by selecting or constructingthe appropriate membrane design, then combining withother bioseparation techniques. For validation, use GFPengineered cells, as well as non-modifi ed cells, in mixturesof pathogenic and non-pathogenic microorganisms.• Correlate media composition to changes in growthcharacteristics and metabolism of L. monocytogenescells, and develop media that enhance the response ofpathogenic cells to detection methods. A complimentaryobjective was to improve low conductivity mediafor the enhanced capture and detection of stressedcells by antibodies and other bioreceptors.• Combine antibody-based capture and growth detectionfor the biochip. Design and microfabricate integrateddevices to combine ATP, pH, and/or direct nucleic acidand antibody-based detection on-chip using multi-channel,multi-functional designs. The goal was to obtain biochipsthat sense multiple parameters simultaneously andimprove dielectrophoresis (DEP)-based selective captureof L. monocytogenes and other pathogens in mixturesof cells and to test sensitivity and selectivity of capture.Project HighlightsWe integrated the multiple functions needed for thedevelopment and deployment of microfl uidic biochips fordetecting bacterial pathogens. We integrated antibody-basedcapture of bacterial cells enhanced by dielectrophoreticforces, bacterial culture and electrical detection of bacterialgrowth, and PCR-based detection of L. monocytogenes—allon chip. A signifi cant accomplishment was the developmentof label-free electrically amplifi ed PCR products. We showedthat impedance can be used to detect the presence of DNAmolecules in a solution without any labels directly fromthe actual PCR solution. This could lead to user-friendlyminiaturized PCR detection systems that do not need opticallabels or optical detectors.“We showed that impedance can be used to detect the presence of DNA moleculesin a solution without any labels directly from the actual PCR solution.”9Center for Food Safety Engineering

Dr. Gehring and Dr. PaoliSince the inception of the Center for Food Safety Engineering, there has been a strong emphasis on interaction and collaboration withscientists from the UDSA-ARS Eastern Regional Research Center. This collaboration has been fostered through interaction betweenscientists during annual Purdue-CFSE/ARS-ERRC research planning workshops, annual participation by Purdue-CFSE and ARS-ERRCscientists in the Molecular Detection Symposium during the International Workshop on Rapid Methods and Automation in Microbiology atKansas State University, and more recently, an invitation for Purdue-CFSE scientists to participate in an ARS-FSIS (Food Safety InspectionService) 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-reviewedjournals.Drs. Andrew Gehring and Arun Bhunia have interacted extensively on their mutual interest in the development of a mixed cultureenrichment (Salmonella, E. coli O157:H7, Listeria monocytogenes, and Yersinia enterocolitica) in support of their work on multiplexedantibody-based microarray for detection of pathogenic bacteria from food. They have worked both independently and collaboratively on thedevelopment of the antibody-based microarray and have co-authored one publication on the subject. As part of her doctoral training, oneof Dr. Bhunia’s doctoral students, Kristen Nanchansky, spent a summer at ERRC working with Dr. Gehring developing a chemiluminescentimmunoassay for the detection of L. monocytogenes. In addition, Drs. Bhunia and Gehring organized and co-chaired a symposium onOptical Technologies for Industrial, Environmental, and Biological Sensing–Food Safety and Agricultural Monitoring for the InternationalSociety for Optical Engineering Photonics East.Drs. George Paoli and Arun Bhunia share an interest in studying the foodborne pathogen Listeria monocytogenes. Dr. Bhunia hasdeveloped and characterized a number of monoclonal antibodies against L. monocytogenes, and Dr. Paoli has applied antibody phagedisplay to select a single-chain antibody specifi c to L. monocytogenes. In addition to co-authoring a chapter on the bacterium in a recentlypublished book on the microbiology and molecular biology of foodborne pathogens, Drs. Paoli and Bhunia collaborated in supervising apost-doctoral study by Dr. Viswaprakash Nanduri on the development of a phage displayed antibody-based surface plasmon resonanceoptical biosensor for the detection of L. monocytogenes.International Collaboration: CFSE goes to ChinaBy invitation of Dr. Xianming Shi, the deputy director of the Bor-Luh Chinese Food Safety Center, Purdue scientists Arun Bhunia andRichard Linton joined USDA-ARS scientists (Shu-I Tu, Andrew Gehring, Yiping He, and George Paoli) and Dr. Jim Lindsay (ARS NationalProgram Leader for Food Safety) in China in May 2008 to meet with representatives from Jiao-Tong University in Shanghai. At the meetingan agreement was signed forming a joint U.S.–Sino Food Safety Research Center between ARS and the Chinese Ministry of Scienceand Technology. We had a very productive meeting and already have plans to work together with USDA-ARS and Jiao-Tong Universityscientists 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 relationshipwith Jaio Tong University in Shanghai, China.Above: A group photograph with our new colleagues and friends fromChina.11Center for Food Safety Engineering

Lu, Bhunia and ChengRapid, quantitative, and reusable immunosensors forbacteria detection on a microfluidic platformInvestigators: Chang Lu (Department of Agricultural and Biological Engineering), Arun Bhunia (Department ofFood Science), Zhongyang Cheng (Materials Engineering, Auburn University)Project RationalePortable, rapid, and sensitive biosensors for food safetyapplications enable point-of-care contamination detectionand immediate interpretation of the results. In our researchproject, we proposed to develop an integrated biosensorsystem on a microfl uidic chip for detecting bacteria basedon immunoassays. The device will offer a sensitivity of 10 2to 10 3 bacteria cell detection and an assay time of fewer than20 minutes for a single test. Our system will yield quantitativedata for estimating the number of the target bacterium in a foodsample. The microfl uidic system will consist of individual devicesfor cell lysis, lysate purifi cation, and immunoassays. In principle,the tool will be effective for any bacterium or strain given theavailability of a suitable intracellular antigen-antibody pair. In thisproject, we will demonstrate the concept using an intracellularantigen, alcohol acetaldehyde dehydrogenase (Aad), and itsantibody MAb-H7 to detect Listeria monocytogenes. In order toconcentrate L. monocytogenes cells from food samples, we willfabricate magnetic nanobars with different sizes and geometriesand develop protocols for immobilizing antibodies specifi c to L.monocytogenes on the surface.A portable, reusable, and low-cost device would be usefulfor point-of-care analysis in the food manufacturing industry.Conducting bacteria detection tests within food manufacturinglaboratories would dramatically decrease the turnaroundtime for the results and avoid potential contamination andchanges in the bacteria during transit. Conventional analyticalmethods require bulky, expensive equipment that are oftencost-prohibitive for food manufacturing laboratories. With ourlab-on-a-chip approach, sophisticated functions of a biologicallaboratory can be miniaturized on a microchip, enabling anyminimally equipped laboratory with the ability to perform bacteriadetection tests. This technology can signifi cantly benefi t the foodindustry by enhancing the laboratory-testing capabilities of foodmanufacturers and food testing laboratories as well as fi eldtestingactivities of governmental agencies.Project Objectives• Fabricate magnetic nanobars with different sizes andgeometries and develop protocols for immobilizingantibodies specifi c to L. monocytogenes on the surface.The amount of bacterial cells bound to the surfacewill be characterized under different conditions.• Develop an electrophoresis-based immunoassaycoupled with laser-induced fl uorescence on a microfl uidicchip. We will use this tool to quantitatively detect L.monocytogenes based on cell lysate via the interactionbetween alcohol acetaldehyde dehydrogenase(Aad) and its monoclonal antibody (MAb-H7).• Demonstrate a prototype-integrated microfl uidicsystem which incorporates different steps suchas manipulation of magnetic nanobars, cell lysis,lysate purifi cation, and immunoassay.Project HighlightsWe integrated the concentration, lysis, and competitiveimmunoassay for detecting L. monocytogenes on a microfl uidicchip. A packed bed of microbeads, with the bead surface coatedwith monoclonal antibody (MAb-H7) that is specifi c to the antigenListeria adhesion protein (LAP), was formed in a microfl uidicchannel to physically trap bacterial cells. Electrical lysis wasthen used to release intracellular materials from the trappedcells. Fluorescein isothiocyanate (FITC)-labeled LAP was fl owedthrough the microbead bed to bind to unreacted antibody sitesand reveal whether LAP was present in the bacterial sample. Byintegrating all these functions onto one simple portable chip, wecan produce a biosensor system that is both highly effi cient andinexpensive.12Center for Food Safety Engineering“By integrating all these functions onto one simple portable chip, we canproduce a biosensor system that is both highly effi cient and inexpensive.”

Nivens, Franklin and CorvalanContinuous monitoring of chemical agents in aqueousmedia using bioreporter-based sensorsInvestigators: David Nivens (Department of Food Science), Michael Franklin (Montana State University), Carlos Corvalan (Department of Food Science)Project RationaleChemical contamination of our food supply threatens the heathof consumers and has become a major concern. As societiesbecome more populated and technologically-advanced, sourcesof pollution and the potential for contamination (inherent,unintentional, or intentional) are increasing. Inexpensivesensors that have analytical capabilities of detecting harmfulchemicals in food would facilitate our nation’s ability to protectits food supply and minimize health concerns associated withcontamination. We proposed to develop bioreporter-basedchemical sensors consisting of genetically programmed cells(bioreporters), a disposable cartridge system containing thebioreporters, and a detection/communication module for Webbasedand/or networked-based assessment capabilities. Withthe successful development of this technology, we anticipate thatthe bioreporter-based chemical sensors will have the analyticalcapabilities required to fi ll a critical need in the food industry.In addition to being potentially inexpensive, these biosensorsare being developed to detect a hazardous chemical belowimmediately dangerous to health of life (IDHL) limits, minimizefalse positives and negatives, have rapid response times, andbe simple to use. We envision that the sensors could be usedwith standard food defense practices to further facilitate a safefood supply.Project Objectives• Develop a dual-signaling bioreporter that minimizesfalse negatives by using bioluminescence andfl uorescence signal for hazardous chemical detection.• Develop a microenvironment that containsprogrammable cells and nutrients to increase thestability and extend the lifetime of the biosensor.• Construct novel bioreporters with optimalanalytical performance for point-of-use andlong-term monitoring experiments.• Model the systems to improve all aspects of analyticalperformance and develop application-specifi cbiosensors for food and agriculture systems.Project HighlightsBioreporter-based chemical sensor technology was used toquantify arsenite concentrations (one of the most hazardousforms of arsenic) in liquid food matrices including milk, fruitjuices, and bottled water with minimal or no sample preparation.For example, various amounts of arsenite were spiked intothe undiluted apple juice samples (arsenite is a commoncontaminate found in apple orchards). An aliquot of eachsample was then exposed to the sensor to generate timedependentlinear calibration curves. Results showed thatapple juice samples with 10 parts per billion (μg/L) arsenitecould be quantifi ed in less than 2 hours. Web- and networkbasedsoftware was developed to monitor the responses of thebioreporter-based sensors and generate a warning signal whenthe analyte concentration exceeded a predetermined alarmlevel. These fi ndings indicate that our technology potentially canbe used by minimally trained food and agricultural workers todetect arsenite (and eventually other contaminates) in a liquidfood matrix at or below chronic and acute minimal risk levels.14Center for Food Safety Engineering“...our technology potentially can be used by minimally trained food and agriculturalworkers to detect arsenite (and eventually other contaminates) in a liquid food matrix...”

Park, Leary and AronsonField-ready biosensors for high throughput andmultiplexed detection of foodborne pathogensInvestigators: Kinam Park (Department of Biomedical Engineering), James Leary (Department of BiologicalEngineering), Arthur Aronson (Department of Biological Sciences)Project RationaleThe increased incidence of pathogen-contaminated food placesa new emphasis on the rapid detection and quantifi cation offoodborne pathogens. Therefore, we are developing a surfaceplasmon resonance (SPR) imaging biosensor for the rapid, labelfree,and high throughput detection of foodborne pathogens. Thisdevice integrates an SPR imaging system with a biosensor arrayimmobilized onto a sample surface containing specifi c shortpeptide ligands. A group of short peptides specifi c to certainpathogenic bacteria will be microcontact-printed on a gold chipin linear patterns. This peptide-imprinted gold chip functions as abiosensor array for the specifi c detection of unknown foodbornepathogens. To determine what fraction of pathogenic bacteriaare live or dead and to confi rm the SPR results, we have createda novel hybrid SPR/molecular imaging portable system.The device would offer a commercial advantage to the foodprocessing industry. It is miniaturized, has fewer components,and is easier to use compared to the current detection systems.This biosensor would detect foodborne pathogens present in

Stanciu and AndreescuPortable biosensor for rapid and ultra-sensitive identificationof organophoshorous foodborne contaminantsInvestigators: Lia Stanciu (Department of Materials Engineering), Silvana Andreescu (Clarkson University)Project RationaleThe overall goal of this project was to advance the fi eld ofpesticide detection in food by developing ultra-sensitivebiosensors based on immobilized acetylcholinesterase (AChE).Over the past decades, AChE biosensors have emerged as apromising technique for food quality control. The developmentof these biosensors could complement or replace classicalanalytical methods by simplifying or eliminating samplepreparation and making identifi cation of chemical foodbornecontaminants easier and faster, with signifi cant decreases inanalysis time and cost.Project Objectives• Establish the optimum parameters for the immobilizationof AChE and the need and feasibility of using anoxidation strategy for phosphorothionates.• Fabricate and characterize the AChE biosensorby immobilizing the enzyme onto the surfaceof single-use screen-printing electrodes (SPE).Study enzyme stability and leaching.• Detect pesticides. Obtain calibration plots of theinhibitory degree upon application of variousconcentrations of pesticides. Determine thedetection limit (DL), response time (RT), and linearconcentration range (LCR) for selected pesticides.• Assemble and test the biosensor prototype for the analysisof pesticides in food samples. Evaluate the matrix effectsand establish whether an extraction step is needed.biosensors with enhanced characteristics. We are the fi rstresearchers to use this direct binding of enzymes onto Ni-NPsfor this purpose. This accomplishment is important becausesite-specifi c orientation of enzymes onto electrode surfaceshas numerous advantages over classical procedures. It ishighly sensitive, avoids conformational changes, decreasessensor costs (due to a lower enzyme requirement), and entailsa simple single fabrication step. This method has the potentialto become a robust, commercially viable system.Alternatively, the procedure combining sol-gel technology withscreen-printing protocols is also potentially useful for biosensorfabrication. Moreover, the sol-gel method is a versatile andeffi cient technique for conserving enzyme activity in organicsolvents. We expect that this matrix will enable functionality ofthe enzyme in the presence of organic solvents, if this mediumis necessary to extract the pesticides from the food matrix.Project HighlightsWe have made signifi cant progress toward establishingthe optimum parameters for enzyme immobilization usingtwo matrices (sol-gel and Ni-nanoparticles)) that enablepreservation of enzymatic activity. The procedure involvingattachment via affi nity binding to Ni-nanoparticles is new andhighly innovative and can be used to fabricate a new class of16Center for Food Safety Engineering“The development of these biosensors could complement or replace classicalanalytical methods by simplifying or eliminating sample preparation and makingidentifi cation of chemical foodborne contaminants easier and faster.”

Scientific Publications and PresentationsPeer Reviewed Journal Publications (2007-2008)• Bae, E., Banada, P.P., Huff, K., Bhunia, A.K., Robinson,J.P., Hirleman, E.D. Analysis of time-resolved scatteringfrom macroscale bacterial colonies. Journal ofBiomedical Optics. 2008. v. 13 (1). p. 014010.• Banerjee, P., Lenz, D., Robinson, J.P., Rickus, J.L., Bhunia,A.K. A novel and simple cell-based detection systemwith collagen-encapsulated B-lymphocyte cell line as abiosensor for rapid detection of pathogens and toxins.Laboratory Investigation. 2008. v. 88. p. 196-206.• Bao, N., Jagadeesan, B., Bhunia, A.K., Yao, Y., Lu,C. Quantifi cation of bacterial cells based onautofl uorescence on a microfluidic platform. Journalof Chromatography. 2008. v. 1181. p. 153-158.• Bao, N., Lu, C. A microfluidic device for physicaltrapping and electrical lysis of bacterial cells. AppliedPhysics Letters. 2008. v. 92. p. 214103.• Bao, N., Wang, J., Lu, C. Recent advances in electricanalysis of cells in microfluidic systems. Analytical andBioanalytical Chemistry. 2008. v. 391. p. 933-942.• Bhattacharya, S., Salamat, S., Morisette, D., Banada, P.,Akin, D., Liu, Y-S., Bhunia, A. K., Ladisch, M., Bashir, R.PCR based-detection in a micro-fabricated platform.Lab on a Chip. 2008. v. 8. p. 1130-1136.• Bhattacharya, S., Jang, J., Yang, L., Akin, D., Bashir, R.BioMEMS and nanotechnology based approaches for rapiddetection of biological entities. Journal of Rapid Methodsand Automation in Microbiology. 2007. v. 15. p. 1-32.• Bhunia, A.K. Biosensors and bio-based methods for theseparation and detection of foodborne pathogens. Advancesin Food and Nutrition Research. 2008. v. 54. p. 1-44.• Burgula, Y., Khali, D., Kim, S., Cousin, M.A., Gore, J. P.,Reuhs, B.L., Mauer, L.J. Review of mid-IR Fouriertransforminfrared (FT-IR) spectroscopy applicationsfor bacterial detection. Journal of Rapid Methods andAutomation in Microbiology. 2007. v. 15. p. 146-175.• Chapple, C., Ladisch, M., Meilan, R. LooseningLignin’s grip on biofuel production. NatureBiotechnology. 2007. v. 25 (7). p. 746-748.• Jedlica, S.S., Little, K.M., Nivens, D.E., Zemlyanov, D., Rickus,J.L. Peptide ormosils as cellular substrates. Journal ofMaterials Chemistry. 2007. v. 17. p. 5058-5067.• Kim, H., Bhunia, A.K. SEL, a selective enrichment broth forsimultaneous growth of Salmonella enterica, Escherichiacoli O157:H7, and Listeria monocytogenes. Applied andEnvironmental Microbiology. 2008. v. 74 (15). p. 4853-4866.• Kim, G., Morgan, M.T., Ess, D.R., Hahm, B.K., Kothapalli, A.,Bhunia, A.K. An automated fiber-optic biosensor based bindinginhibition assay for the detection of Listeria monocytogenes.Food Science and Biotechnology. 2007. v. 16(3). p. 337-342.• Kim, Y., Hendrickson, R., Mosier, N. S., Ladisch, M. R., Bals,B., Balan, V., Dale, B.E. Enzyme hydrolysis and ethanolfermentation of liquid hot water and afex pretreateddistillers’ grains at high-solids loadings. BioresourceTechnology. 2008. v. 99(12). p. 5206-5215.• Kim, Y., Mosier, N. S., Hendrickson, R., Ezeji, T., Blaschek, H., Dien,B., Cotta, M., Dale, B., Ladisch M. R. Composition of corn drygrindethanol by-products: DDGS, 3 wet cake, and thin stillage.Bioresource Technology. 2008. v. 99(12). p. 5165-5176.• Kim, Y., Mosier, N., Ladisch, M. R. Process simulationof modified dry grind ethanol plant with recycle ofpretreated and enzymatically hydrolyzed distillers’ grains.Bioresource Technology. 2008. v. 99(12). p. 5177-5192.• Ladisch, M., Dale, B., Tyner, W., Mosier, N.S., Kim, Y., Cotta, M.,Dien, B.S., Blaschek, H., Laurenas, E., Shanks, B., Verkade, J.,Schell, C., Petersen, G. Cellulose conversion in dry grind ethanolplants. Bioresource Technology. 2008. v. 99(12). p. 5157-5159.• Ladisch, M. R., Dale, D. Distillers grains: On thepathway to cellulose conversion. BioresourceTechnology. 2008. v. 99(12) p. 5155-5156.• Lathrop, A.L., Banada, P.P., Bhunia, A.K. Differentialexpression of InlB and ActA in Listeria monocytogenesin selective and nonselective enrichment broths. Journalof Applied Microbiology. 2008. v. 104. p. 627-639.• Liu, Y-S., Banada, P.P., Bhattacharya, S., Bhunia, A.K.,Bashir, R. Electrical characterization of DNA moleculesin solution using impedance measurements. AppliedPhysics Letters. 2008. v. 92. p. 143902.• Liu, Y-S., Walter, T. M., Chang, W-J., Lim, K-S., Yang, L.,Lee, S-W., Aronson, A., Bashir, R. Electrical detectionof germination of model Bacillus Anthracis spores inmicrofluidic biochips. Lab Chip. 2007. v. 7. p. 603-610.• Stewart, P.S., Franklin, M.J. Physiological heterogeneity inbiofilms. Nature Reviews Microbiology. 2008. v. 6 p.199-210.• Vermerris, W., Saballos, A., Ejeta, G., Mosier, N. S., Ladisch,M. R., Carpita, N. C. Molecular breeding to enhance ethanolproduction from corn and sorghum stover. Crop ScienceSociety of America. 2007. v. 47(S3). p. S142-S153.• Wang, C., Irudayaraj, J. Gold nanorod probesdetects multiple pathogens. Small – aNanotechnology Journal. 2008. (In Press).• Ximenes, E. A., Dien, B. S., Ladisch, M. R., Mosier, N.,Cotta, M. A., Li, X. L. Enzyme production by industriallyrelevant fungi cultured on feed co-product from corndry grind ethanol plants. Applied Biochemistry andBiotechnology. 2007. v. 136-140 (1-12). p. 171-183.• Yang, L., Banda, P.P., Bhunia, A.K., Bashir, R. Effects ofdielectrophoresis on growth, viability, and immunoreactivityof Listeria monocytogenes. Journal ofBiological Engineering. (2008) v. 2(6). p.“This year, the CFSE team published 31 peer-reviewed researchpublications and presented 18 talks at national science meetings.”17Center for Food Safety Engineering

Scientific Publications and Presentations• Yang, L., Bashir, R. Electrical/electrochemicalimpedance for rapid detection of foodbornepathogenic bacteria. Biotechnology Progress,Biotechnology Advances. 2008. v. 26. p. 135-150.• Yu, C., Irudayaraj, J. Multiplex biosensor using gold nanorods.Analytical Chemistry. 2007. v. 79(2) p. 572-579.• Yu, C., Irudayaraj, J. Sensitivity and selectivitylimits of multiplex nanoSPR biosensor assays.Biophysical Journal. 2007. v. 93(9) p.1-9.• Zeng, M. N., Mosier, S., Huang, C-P., Sherman, D. M.,Ladisch, M. R. Microscopic examination of changesof plant cell structure in corn stover due to cellulaseactivity and hot water pretreatment. BiotechnologyBioengineerg. 2007. v. 97(2). p. 265-278.Abstracts for Major Papers/Posters Presented(2007-2008)• Banada, P.P., Bernas, T., Robinson, J.P., Bhunia, A. K.Proteomic analysis of cytotoxic factors from Bacilluscereus. American Society for Microbiology GeneralMeeting. Toronto, ON. May 21-25, 2007.• Bashir, R., Ahmad, I. BioMEMS and bionanotechnologyfor development of miniaturized instruments.Symposium on Center for Analytical InstrumentationDevelopment. West Lafayette, IN. June 18, 2008.• Bashir, R., Bhunia, A., Ladisch, M. Engineering of biosystemsfor the detection of Listeria monocytogenes in foods—development of a biochip. Manhattan, KS. June 18, 2008.• Bashir, R. Interfacing Silicon and Biology at theMicro and Nanoscale. NSF USA-EU Workshop onBionanotechnology. Ispra, Italy. May, 2008.• Bashir, R. Interfacing silicon and biology at the micro andnanoscale. University of Cincinnati, Nanomedicine CenterSeminar Series. Cincinnati, OH. February 18, 2008.• Bashir, R. BioMEMS and bionanotechnology: Integration oflife sciences and engineering at the micro and nanoscale.The Knowledge Foundation’s 10 th Annual Conference,BioDetection Technologies. Atlanta, GA. June 14-15, 2007.• Bhattacharya, S., Salamat, S., Banada, P., Liu, Y.,Morisette, D., Bhunia, A. K., Akin, D., Bashir, R. Integrateddetection of microorganisms in a microfluidic biochip.Biomedical Engineering Society (BMES) AnnualMeeting. Los Angeles, CA. September 27-29, 2007.• Burkholder, K. M., Kim, K.-P., Hahm, B.-K., Mishra, K., Medina-Maldonado, S., Bhunia, A. K. Anaerobic environmentincreases surface localization of Listeria AdhesionProtein (LAP) and promotes infectivity of Listeriamonocytogenes. American Society for MicrobiologyGeneral Meeting. Boston, MA. June 1-5, 2008.• Jagadeesan, B., Raizman, E., Nanduri, V., Bannantine, J., Bhunia,A.K. Rapid label free sero-diagnosis of Johne’s disease usingsurface plasmon resonance biosensor. American Society forMicrobiology General Meeting. Boston, MA. June 1-5, 2008.• Kim, K., Bhunia, A.K. Performance evaluation of a multiplexselective enrichment broth, SEL, by proteomic analysisand immunoassay. Institute of Food Technologist AnnualMeeting. New Orleans, LA. June 29-July 2, 2008.• Koo, O. K., Shuaib, S., Ladisch, M. R., Bashir, R., Bhunia,A. K. Targeted capture of pathogenic Listeria using aListeria Adhesion Protein (LAP) specific mammaliancell receptor, Hsp60 for detection of bacteria onbiosensor platforms. American Society for MicrobiologyGeneral Meeting. Boston, MA. June 1-5, 2008.• Koo, O. K., Jagadeesan, B., Burkholder, K., Bhunia, A.K.Targeted capture of pathogenic Listeria using a ListeriaAdhesion Protein (LAP) specific mammalian cellreceptor, Hsp60 for detection of bacteria on biosensorplatforms. Institute of Food Technologists AnnualMeeting. Chicago, IL. July 28-August 1, 2007.• Lenz, A.P., Williamson, K., Franklin M.J. Localized geneexpression along vertical transects of Pseudomonasaeruginosa biofilms. American Society for MicrobiologyMeeting on Microbial Biofilms. 2008.• Lenz, A.P., Williamson K., Franklin M.J. Quantification of cellnumbers and riboscome content of Pseudomonas aeruginosabiofilms using laser microdissection and qRT-PCR. GeneralMeeting of the American Society for Microbiology. 2008.• Liu, Y., Banada, P., Bhattacharya, S., Akin, D., Bhunia,A. K., Bashir, R. Electrical characterization of DNAmolecules in fluids using impedance measurements.Biomedical Engineering Society (BMES) AnnualMeeting, Los Angeles, CA. September 27-29, 2007.• Mishra, K., Burkholder, K. M., Medina-Maldonado, S., Bhunia,A. K. Cloning, genomic organization and expression ofSecA2 Gene in Listeria species. American Society forMicrobiology General Meeting. Boston, MA. June 1-5, 2008.• Morandage, J. S., Woloshuk, C. P., Cousin, M. A. Releaseof DNA from Fusarium spores for use in real-time PCR.Institute of Food Technologists. 2008. Abstract p.• Morandage, J. S., Woloshuk, C. P., Cousin, M. A.Enzymatic release of DNA from Fusarium sporesfor use in real-time PCR. International Associationfor Food Protection. 2008. Abstract p.• Nagel, A.C., Schroeder, D.L., Gross, B.D., Co, B., Nivens, D.E.Development of an arsenic sensing biosensor as a model fordetection of chemical threat agents in food products. Instituteof Food Technologist National Meeting. Chicago, IL. 2007.18Center for Food Safety Engineering“At the Center for Food Safety Engineering, we direct our

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

Center StaffDr. Arun K. BhuniaPI765.494.5443bhunia@purdue.eduDr. Maribeth A. CousinPI765.494.8287cousin@purdue.eduCo-PIsAmornrat Aroonnual • 765.496.3826 • aaroonnu@purdue.eduEuiwon Bae • 765.494.4762 • ebae@purdue.eduAndrew Gehring • 215.233.6491 • andrew.gehring@ars.usda.govE. Daniel Hirleman • 765.494.5688 • hirleman@purdue.eduSeung Ohk • 765.496.7356 • sohk@purdue.eduValery Patsekin • 765.494.0757 • vpatseki@purdue.eduBartek Rajwa • 765.494.0757 • rajwa@fl owcyt.cyto.purdue.eduJ. Paul Robinson • 765.494.6449 • jpr@fl ocyt.cyto.purdue.eduShu-I-Tu • 215.233.6466 • shui.tu@ars.usda.govStaff / Graduate StudentsNan Bai • nbai@purdue.eduAmanda Bettasso • bettasso@purdue.eduHyochin Kim • 765.496.7354 • kin399@purdue.eduCo-PICharles P. Woloshuk • 765.494.3450 • woloshuk@purdue.eduStaff / Graduate StudentsYenny Suanthie • ysuanthi@purdue.eduJanaka Morandage • jmoranpa@purdue.eduDr. Joseph IrudayarajPI765.494.0388josephi@purdue.eduDr. Chang LuPI765.494.1188changlu@purdue.eduDr. Mark MorganPI765.494.1180mmorgan@purdue.eduCo-PIsChitrita DebRoy • rcd3@psu.eduPIna Fratamico • pina.fratamico@ars.usda.govLisa Mauer • 765.494.9111 • mauer@purdue.eduStaff / Graduate StudentsDeepali Herlekar • dah1001@psu.eduSandeep Ravindran • psandeep@purdue.eduChungang Wang • 765.494.0388 • wang1032@purdue.eduCo-PIsNing Bao • nbao@purdue.eduArun Bhunia • 765.494.5443 • bhunia@purdue.eduZhongyang Cheng • chengzh@auburn.eduStaff / Graduate StudentsHsiang-Yu Wang • hwang@purdue.eduBalamurugan Jagadessan • bala@purdue.eduPeixuan Wu • wupeixu@auburn.eduCo-PIBruce Applegate • 765.496.7920 • applegate@purdue.eduStaffLynda Perry • 765.494.7698 • llperry@purdue.edu20Center for Food Safety Engineering

Dr. Michael LadischPI765.494.7022ladisch@purdue.eduDr. David E. NivensPI765.494.0460dnivens@purdue.eduDr. Kinam ParkPI765.494.7759kpark@purdue.eduDr. Lia StanciuPI765.496.3552lstanciu@purdue.eduCo-PIsAmornrat Aroonnual • 765.496.3826 • aaroonnu@purdue.eduRashid Bashir • bashir@purdue.eduArun Bhunia • 765.494.5443 • bhunia@purdue.eduYoungmi Kim • kim107@purdue.eduXingya Liu • 765.494.-7052 • xingya@purdue.eduKrishna Mishra • 765.494.6236 • kmishra@purdue.eduNathan Mosier • 765.494.7025 • mosiern@purdue.eduJ. Paul Robinson • 765.494.6449 • joseph.p.robinson.1@purdue.eduAndres Rodriguez • arodriguez1976@gmail.comEduardo Ximenes • eximenes@purdue.eduMiroslav Sedlak • 765.494.3699 • sedlak@purdue.eduStaff / Graduate StudentsKristin Burkholder • 765.496.7354 • kburkhol@purdue.eduBala Jagadeesan • 765.496.7356 • bjagadee@purdue.eduOk Kyung Koo • 765.496.7354 • okoo@purdue.eduYi-Shao Liu • liu63@purdue.eduJaeho Shin • shin0@purdue.eduDavid Sung • ysung@purdue.eduShuaib Salamat • ssalamat@purdue.eduStefan Schwietzke • sschwiet@purdue.eduAngela Valadez • 765.496.3824 • valadez@purdue.eduCo-PIsCarlos Corvalan • 765.494.8262 • corvalac@purdue.eduMichael Franklin • 406.994.5658 • umbfm@montana.eduStaff / Graduate StudentsBen Gross • 765.494.6960 • bdgross@purdue.eduClaudia Ionita • 765.496.7354 • cionita@purdue.eduAilyn Lenz • alenz@monana.eduAaron Nagel • 765.496.3832 • acnagel@purdue.eduCo-PIsJames F. Leary • 765.494.7280 • jfl eary@purdue.eduArthur I. Aronson • aronson@purdue.eduGhanashyam Acharya • gacharya@purdue.eduJong-Ho Kim • kim606@purdue.eduStaff / Graduate StudentsMeggie Grafton • 765.494.2955 • mgrafton@purdue.eduMichael Zordan • mzordan@purdue.eduCo-PIDr. Silvana Andreescu • 315.268.2394 • eandrees@clarkson.eduStaff / Graduate StudentsMallikarjunarao Ganesana • 315.368.3806 • ganesanm@clarkson.eduBrian Frederick • 315.368.2348 • frederbj@clarkson.edu21Center for Food Safety Engineering

It is the policy of the Purdue University Center for Food Safety Engineering, that all persons shall have equalopportunity 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 Affirmative Action employer.The Center for Food Safety EngineeringPurdue UniversityFood Science Building745 Agriculture Mall DriveWest Lafayette, IN 47909Non-profitOrganizationU. PostagePAIDPurdueUniversity