Progress 09/01/03 to 08/31/05
Outputs
This project has two objectives, namely: (1) to evaluate the performance of the electrochemical biosensor in varying concentrations of the polyaniline and varying types and concentrations of antibodies and (2) to validate the redesigned biosensor for detecting various isolates of Escherichia coli O157:H7 and Salmonella species as model pathogens in pure culture and in selected artificially-contaminated fresh produce samples. Detailed accomplishments are described below. Objective 1: Polyaniline (Pani) nanowires were successfully synthesized from three types of acids. The purpose was to determine which protonating acid would result in the most conductive nanowire for the subsequent fabrication of the biosensor. The acids were 4-hydroxybenzenesulfonic acid, phenylphosphonic acid, 4-sulfobenzoic acid, and hydrochloride acid (HCl). A scanning electron microscope and a transmission electron microscope were used to visualize the Pani nanowires. Polyaniline was also
successfully conjugated with antibodies to form the molecular bio-wire for the biosensor. We evaluated monoclonal and polyclonal antibodies in this biosensor platform. The rationale for not having a combination of polyclonal on the conjugate pad and monoclonal on the capture pad was the possibility that the polyclonal antibodies might saturate the binding sites of the antigen surface, leaving no open site for binding with the monoclonal antibodies that were immobilized on the capture pad. This deficiency would prevent the sandwich effect that was needed for the conductive polyaniline to make a circuit and release an electrical signal. The final fabricated biosensor was disposable, sensitive, specific, and reagentless. The biosensor dimensions were 5 mm wide, 2 mm thick, and 70 mm long. Objective 2: The fabricated biosensor was then used to detect generic E. coli, E. coli O157:H7, Salmonella Typhimurium, Salmonella Thompson, and Salmonella Newport in serially diluted cultures and
artificially contaminated fruits and vegetables. Selected fruits and vegetables were artificially inoculated with cell cultures. The detection process, from sample application to output readout, took between 2 and 6 minutes. Data on the detection of E. coli O157:H7 and Salmonella showed that the lower limit of detection was 80-100 colony forming units (cfu) per ml. The linear range of effective enumeration was between 100 and 100,000 cfu per ml while the effective range of detection was between 100 and 100 million cfu per ml. The E. coli O157:H7 antibodies used in the study were specific to E. coli O157:H7 organisms while the generic E. coli antibodies were reactive to all E. coli strains, including E. coli O157:H7. The biosensors prepared with anti- E. coli O157:H7 did not respond to the presence of E. coli K-12 and Salmonella Typhimurium, which were added to the inoculated samples, demonstrating specificity of the antibodies used. In summary, the major accomplishments of this
project were the successful fabrication of disposable biosensor units for the rapid and sensitive detection of E. coli O157:H7 and Salmonella species and their validation in selected fresh produce samples.
Impacts
Although the US food supply is unmatched in quality and quantity, we continue to face new challenges involving food safety. Biological threats of the food supply chain and water systems from terrorists are also a reality. Furthermore, novel pathogens are emerging; familiar ones are growing resistant to antibiotic treatment. Food production and processing are increasingly becoming centralized. Americans eat in restaurants more and we eat more imported foods, some of which come from across the globe virtually overnight. These changes require strengthened systems of pathogen monitoring. Research on the development of biosensors for the rapid detection of foodborne pathogens is not only important but it is necessary to maintain the integrity and quality of the food chain, to help minimize contaminated products from leaving the processing environment, and to eliminate the microbial contaminants from reaching the dinner table. With threats of bioterrorism becoming even more
intense, the development of onsite detection methods, such as this biosensor can provide, is a requirement for biosecurity. Additionally, this biosensor can potentially reduce the cost of food testing and pathogen diagnostics.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Objective 1: To evaluate the performance of the electrochemical biosensors in varying concentrations of the polyaniline and varying types and concentrations of antibodies. Polyaniline was successfully synthesized as a conductive molecular wire. Scanning electron microscope and transmission electron microscope were used to visualize the nanowires. Polyaniline was successfully conjugated with antibodies to form a molecular bio-wire for the conductometric biosensor. The fabricated biosensor was disposable, sensitive, specific, and reagentless. The biosensor dimensions are: 5 mm wide, 2 mm thick, and 70 mm long. Objective 2: To validate the redesigned biosensors for detecting various isolates of Escherichia coli O157:H7 and Salmonella species as model pathogens in pure culture and in selected artificially-contaminated fresh produce samples. The biosensor was initially tested in pure culture of E. coli O157:H7, Salmonella Typhimurium, Salmonella Thompson, and Salmonella
Newport. The biosensor was also initially used to detect these organisms in selected artificially contaminated fresh produce. Initial results showed that the lower limit of detection of the biosensor was 80-100 cfu/ml. Students: One PhD student and one undergraduate student are being funded by the project. Patent: One patent application has been filed and processed. Peer-reviewed Publications: We published 3 peer-reviewed papers and made 4 presentations in professional meetings in 2003 and 2004.
Impacts
Although the US food supply is unmatched in quality and quantity, we face new challenges involving food safety in the 21st century. Biological threats of the food supply chain and water systems from terrorists are a reality. Furthermore, novel pathogens are emerging; familiar ones are growing resistant to antibiotic treatment. Food production and processing are increasingly becoming centralized. Americans eat in restaurants more and we eat more imported foods, some of which come from across the globe virtually overnight. These changes require strengthened systems of pathogen monitoring. Research on the development of biosensors for the rapid detection of foodborne pathogens is not only important but it is necessary to maintain the integrity and quality of the food chain, to help minimize contaminated products from leaving the processing environment, and to eliminate the microbial contaminants from reaching the dinner table. With threats of bioterrorism becoming even
more intense, the development of onsite detection methods, such as this biosensor can provide, is a requirement for biosecurity. Additionally, this biosensor can potentially reduce the cost of food testing and pathogen diagnostics.
Publications
- List of Peer-Reviewed Publications in 2003 and 2004:
- Muhammad-Tahir, Z. and Alocilja, E.C. 2004. A Disposable Biosensor for Pathogen Detection in Fresh Produce Samples. Biosystems Engineering Journal, 88(2):145-151
- Muhammad-Tahir, Z. and Alocilja, E.C. 2003. Fabrication of a disposable biosensor for Escherichia coli O157:H7 detection. IEEE Sensors Journal, 3(4):345-351.
- Muhammad-Tahir, Z. and Alocilja, E.C. 2003. A conductometric biosensor for biosecurity. Biosensors and Bioelectronics Journal, 18(5-6): 813-819.
- List of Presentations in 2003 and 2004:
- McGraw, S., Muhammad-Tahir, Z., and Alocilja, E.C. 2004. Us of a conductometric biosensor for detection of foodborne pathogens. Poster for the Annual Engineering Summer Research Internship Poster Session, Michigan State University, Oct. 22, 2004.
- Muhammad-Tahir, Z. and Alocilja, E.C. 2004. Preparation of Polyaniline in Various Acids and its Biosensor Application, Poster presentation at the 8th World Congress on Biosensors, Granada, Spain, May 24-26, 2004.
- Alocilja, E.C. and Muhammad-Tahir, Z. 2004. Molecular wires in biosensor development. 2004 Institute of Biological Engineering Annual Meeting, Fayetville, Arkansas, January 9-11, 2004.
- Alocilja, E.C. 2003. Biosensor Development for Rapid Pathogen Detection, National Food Safety and Toxicology Center seminar series, Michigan State University, East Lansing, MI, November 3, 2003.
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Progress 09/01/03 to 12/31/03
Outputs
Funding for this project commenced on September 1, 2003. A graduate student assistant was hired to help conduct the experiments. Supplies were purchased and necessary equipment were put together. As scheduled, we have initially fabricated the biosensor units that we will be using in the subsequent experiments.
Impacts
Although the US food supply is unmatched in quality and quantity, we face new challenges involving food safety in the 21st century. Biological threats of the food supply chain and water systems from terrorists are a reality. Furthermore, novel pathogens are emerging; familiar ones are growing resistant to antibiotic treatment. Food production and processing are increasingly becoming centralized. Americans eat in restaurants more and we eat more imported foods, some of which come from across the globe virtually overnight. These changes require strengthened systems of pathogen monitoring. Research on the development of biosensors for the rapid detection of foodborne pathogens is not only important but it is necessary to maintain the integrity and quality of the food chain, to help minimize contaminated products from leaving the processing environment, and to eliminate the microbial contaminants from reaching the dinner table. With threats of bioterrorism becoming even
more intense, the development of onsite detection methods, such as this biosensor can provide, is a requirement for biosecurity. Additionally, this biosensor can potentially reduce the cost of food testing and pathogen diagnostics.
Publications
- No publications reported this period
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