A Nanostructured Biosensor for the Detection of Microbial Pathogens

NON-TECHNICAL SUMMARY: The rapid and specific detection of foodborne pathogens is paramount to food safety We propose to fabricate a nanostructured bacteriophage-amplified photonic biosensor for the detection of the foodborne pathogen Escherichia coli O157:H7. <P>

APPROACH: This research effort will consist of four essential elements: 1) A deterministically grown nanofiber array of forests and cages will be synthesized on an integrated circuit microluminometer platform. 2) A recombinant reporter phage specific for E. coli O157:H7 will be attached to the nanofiber structures via antibody or avidin linkages. 3) A luxCDABE-based bioluminescent bioreporter will be dispensed in the nanofiber cages via reagent jet deposition. 4) The newly created nanostructured biosensor will be exposed to E. coli O157:H7. Recombinant reporter phage will infect E. coli O157:H7 target cells and instigate the synthesis of signature quorum sensing autoinducer molecules that are then detected by the caged bioluminescent bioreporter cells. The integrated circuit microluminometer measures and monitors bioluminescence emission<P>

PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: This research effort centered on the development of a nanostructured bacteriophage-amplified photonic biosensor for the detection of foodborne pathogens. Although not fully completed under this one-year funding schedule, the foundation for a new biosensor product technology has been established. Novel techniques for attaching bacteriophage to nanofiber elements via antibodies and new on-chip electroosmotic deposition methods were developed. Dissemination of this biosensor platform technology was achieved through invited oral presentations at Pittcon 2007 (Chicago, IL) and at the 234th American Chemical Society National Meeting (Boston, MA). An updated invited oral presentation will be given at the upcoming Pittcon 2008 meeting in New Orleans, LA. The working group of this project additionally presented research findings at the Oak Ridge National Laboratory Center for Nanophase Materials Sciences User's Forum in Oak Ridge, TN. An undergraduate student from the University of Tennessee Department of Microbiology also participated in the project. <BR> PARTICIPANTS: Steven A. Ripp, Ph.D. (PI; 5% effort; The University of Tennessee) Responsible for overall supervision of project and successful integration of the University of Tennessee and Oak Ridge National Laboratory research teams. Managed and set timelines, arranged bi-monthly meetings, and ensured timely submission of annual and final reports. Mitchel J. Doktycz, Ph.D. (co-PI; 5% effort; Oak Ridge National Laboratory) Dr. Doktycz was responsible for overseeing the patterned synthesis of the nanofiber arrays, piezo-deposition of the bioreporters, and sealing of nanostructures. Gary S. Sayler, Ph.D. (co-PI; 5% effort; The University of Tennessee) Dr. Sayler was available for consultation during all aspects of this study. Anatoli V. Meleshko, Ph.D. (co-PI; 5% effort; Oak Ridge National Laboratory) Dr. Meleshko assisted in the growth of the nanofibers on the BBIC microluminometer chip. Jennifer R. Brigati, Ph.D. (Postdoctoral Scientist; 100% effort; The University of Tennessee) Dr. Brigati initially led the experimental research phases of this study and developed methods for phage attachment to nanofibers. James T. Fleming, Ph.D. (Research Scientist; 100% effort; The University of Tennessee) Dr. Fleming replaced Dr. Brigati when she acquired a teaching position at Maryville College, Maryville, TN. He instituted the application of on-chip electroosmotic deposition for target concentration. Lindy Pryer, Undergraduate student, The University of Tennessee Ms. Pryer participated in all aspects of this research effort during her four semesters of independent research. Training and professional development was provided to Lindy Pryer, an undergraduate student in the Department of Microbiology at The University of Tennessee Partner organizations included the Oak Ridge National Laboratory Center for Nanophase Materials Sciences <BR> TARGET AUDIENCES: Laboratory instruction was provided to a University of Tennessee undergraduate student in the Department of Microbiology The final target audience for this research effort is the food safety industry for provision of enhanced monitoring assays for foodborne pathogen detection. <BR> PROJECT MODIFICATIONS: Major changes to the research effort occurred due to our inability to concentrate sufficient bacterial targets (E. coli O157:H7) onto the lab-on-a-chip interface. Thus was instituted techniques related to electroosmotic deposition and a reconfiguring of the nanointerface surface to accommodate additional electrodes.
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IMPACT: 2007/01 TO 2007/12<BR>
Escherichia coli and other foodborne pathogens such as Salmonella, Clostridium, and Listeria are an ever-present hindrance to the food industry, being responsible for several recent major food recalls. The need for faster and more sensitive detection methods is therefore always a priority. Our pre-development of a nanofabricated pathogen sensor module addresses this demand, and may provide the necessary size, portability, and deployability required by the food industry to move quality control testing away from centralized laboratories and more towards the production line itself. The primary objective of this research program was the fabrication of a nanostructured bacteriophage-amplified photonic biosensor for the detection of foodborne pathogens (using E. coli O157:H7 as a model). We have previously demonstrated the feasibility of bacteriophage-based bioluminescent sensing techniques and have reported on a bioluminescent bioreporter integrated circuit (BBIC) microluminometer for standalone detection of bioluminescent signals as well as new piezo-based ink jet deposition methods for defined placement of living cells within highly patterned nanofiber microstructure assemblies. In this effort, each of these technologies was combined to form the final biosensor element. However, E. coli detection limits were substandard due to the inability to concentrate target bacterial cells on the biosensor. This knowledge change instituted new application of on-chip electroosmotic deposition for target concentration. As well, the nanofiber caging of bioreporter cells was deemed unnecessary and an improved on-chip biofilm method was developed for extended lifetime maintenance of bioreporter cells. Techniques to attach reporter bacteriophage to nanofibers were additionally developed. Each of these new components are now being incorporated into the biosensor array and preliminary testing of detection limits after E. coli O157:H7 exposures will soon be performed. The success of this project will result in a change in conditions of current pathogen sensing technologies to yield a miniaturized autonomous biosensor that is easy to use, cost-effective, sensitive, and specific. This will be of significant impact to the food and farming industry.