The overall goal of this proposed research is to develop immuno-electrochemical/optical biosensors for rapid detection of several pathogenic bacteria in poultry, meat and vegetables products. <BR> <BR> The supporting objectives are: <BR> Phase I: Develop immuno-electrochemical and optical biosensing methods based on the immuno-microbeads or capillary-column-based immuno-separator for separation of target bacteria from food samples, microbioreactors for enzymatic amplification, and microelectrodes and optical detectors for signal measurement. Optimize the conditions of antibody immobilization, bacterial separation, enzymatic amplification, sample flow rate, buffer solution, incubation time, blockage reagents, substrate solution, and signal amplification. <BR> <BR> Phase II: Design and fabricate prototype biosensors based on the biosensing methods to be developed in Phase I, by assembly the components of sample pretreatment, biosensing devices and electrochemical/optical transducer into an automated instrument. <BR> <BR> Phase III: Evaluate the biosensors for detection of Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 in raw and cooked poultry and meat products such as chicken carcasses, chicken patties, hamburgers, and hotdogs, and fresh vegetables to demonstrate the applications.
NON-TECHNICAL SUMMARY: To ensure food safety, the food industry needs rapid methods for detection of pathogenic bacteria. Biologically based sensors will be developed for rapid detection of Salmonella, Listeria and E. coli in poultry, meat and vegetables. A prototype biosensor will be designed and fabricated for its applications.
APPROACH: In Phase I, immuno-electrochemical and optical biosensing methods will be investigated based on the immuno-microbeads or capillary-column-based immuno-separator for separation of target bacteria from food samples, microbioreactors for enzymatic amplification, and microelectrodes and optical detectors for signal measurement. Parameters will be determined for the immuno-micromagnetic beads separation, bioseparator/bioreactor, enzymatic reaction, microelectrode and optical detector. The conditions of antibody immobilization, bacterial separation, enzymatic amplification, sample flow rate, buffer solution, incubation time, blockage reagents, substrate solution, and signal amplification will be tested and optimized to minimize the noise level and costs, maximize the signals. In Phase II, prototype biosensors will be designed and fabricated based on the biosensing methods to be developed in Phase I. The components of sample pretreatment, biosensing devices and electrochemical/optical transducer will be assembly into an automated instrument. The calibration lines will be developed for the prototype instrument. Specifications of the biosensor will be determined. In Phase III, the biosensors will be evaluated for detection of Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 in raw and cooked poultry and meat products such as chicken carcasses, chicken patties, hamburgers, and hotdogs, and fresh vegetables to demonstrate the applications. The detection limit and detection time will be determined for each type of food products.
PROGRESS: 2001/10 TO 2007/09<BR>
OUTPUTS: A Quartz Crystal Microbalance (QCM) DNA sensor, based on the nanoparticle amplification, was developed for rapid detection of E. coli O157:H7. A thiolated single stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the frequency change of the QCM. Streptavidin conjugated Fe3O4 nanoparticles were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10-12 M synthesized oligonucleotides and 102 CFU/ml E. coli O157:H7 cells can be detected by the sensor. At the same time, the same QCM biosensor was investigated to directly detect the cells of E. coli O157:H7 captured by the antibody immobilized on the gold electrode surface with the magnetic nanobeads to amplify the signals in measurement of both frequency and resistance. The result showed that the QCM biosensor could detect E. coli O157:H7 as low as 100 cells/ml in just one to two hours. The immunoseparation with magnetic nanoparticle-antibody conjugates (MNCs) was investigated and evaluated for separating and concentrating target pathogens including E. coli O157:H7, S. Typhimurium and L. monocytogenes in food samples. MNCs were prepared by immobilizing biotin labeled specific antibodies onto streptavidin-coated magnetic nanoparticles. MNCs were mixed with inoculated food samples, and then nanoparticle-antibody-bacteria complexes were separated from food matrix with a magnet. MNCs presented a minimum CE of 94% for E. coli O157:H7 ranging from 101 to 107 CFU/ml in 15 min without any enrichment. Capture of E. coli O157:H7 by MNCs was not interfered with other bacteria. The use of semiconductor quantum dots (QDs) as fluorescence labels in immunosensors was studied for simultaneous detection of E. coli O157:H7, S. Typhimurium and L. monocytogenes. QDs with emission wavelengths of 525 nm, 610 nm and 705 nm were conjugated to three different antibodies, respectively. Target bacteria were separated from samples using the antibodies coated magnetic microbeads. The bead-cell complexes reacted with QDs-antibody conjugates to form bead-cell-QDs complexes. The intensities of fluorescence emission peaks at three wavelengthes were measured for numbers of three target bacteria. The detection limit was 104 cfu/ml and the detection time was 2 h. This result was published for detection of E. coli and Salmonella, for detection of L. monocytogenes, and for all three types of bacteria with a detection limit of <10 cells/ml within 2 h Impedance biosensor was developed based on microfluidic chips with interdigitaed array microelectrode and magnetic nanobeads immunoseparation for detection of E. coli O157:H7 and L. monocytogenes in food samples. Microfluidics based optical (absorbance and chemiluminescence) biosensors were also investigated for detection of foodborne pathogens. <BR> PARTICIPANTS: The project provided a great opportunity for graduate students' training. Two MS students and one PhD student were involved in this project and have completed their graduate programs. <BR> TARGET AUDIENCES: This project provided researchers in biosensors and people in the area of food safety, specifically with food industries, an updated new biosensor technology for rapid detection of foodborne pathogens.
IMPACT: 2001/10 TO 2007/09<BR>
Contaminated food is estimated to cause 76 million illnesses, 325,000 serious illnesses resulting in hospitalization, and 5,000 deaths in the United States each year (CDC, 1999). The economic impact of foodborne illness has been estimated as high as $10 billion annually (USDA/ERS, 2002). Current practices for preventing foodborne diseases due to microbial contamination of food products rely upon rapid identification and effective control of specific pathogens from farm to fork. However, conventional culture methods are extremely time-consuming, typically requiring at least 24 h and complicated multi-steps to confirm the analysis. Even current rapid methods such as ELISA and PCR still take 4-8 h to generate only qualitative results and require laboratory setup and skilled personnel. Threfore, it is urgently needed by the food industry, agencies and consumers for rapid, sensitive and sepcific detection of foodborne pathogens in food products, specifically ready-to-eat foods. The biosensors that have been developed in this project will provide the food industry with more rapid, specific, sensitive and cost-effective method for the detection of pathogenic bacteria in food products. This research will lead to the development of portable biosensor instruments for rapid detection of foodborne pathogens for on-line or in-field applications. Therefore, the outcome of this study on biosensors for rapid detection of foodborne pathogens will assist the food industry in their enhancing HACCP programs to ensure food safety and security, which will reduce foodborne diseases and minimize food product recalls associated with microbial contamination. The biosensors can also be applied to other areas such as environmental protection, clinical diagnosis and antibioterrorists in homeland security.