1. A Membrane Strip Immunosensor for Foodborne Pathogen Detection Zarini Muhammad-Tahir Evangelyn C. Alocilja Michigan State University ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

2. Outline IntroductionIntroduction Methods and materialsMethods and materials ResultsResults DiscussionDiscussion ConclusionConclusion ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

3. Introduction 90% of foodborne illnesses caused by bacteria90% of foodborne illnesses caused by bacteria Escherichia coli O157:H7 and Salmonella typhimuriumEscherichia coli O157:H7 and Salmonella typhimurium Illnesses mild, watery diarrhea, to life-threatening conditions Fatal children, elderly, and immune-compromised individuals Outbreaks related to fresh fruits and vegetablesOutbreaks related to fresh fruits and vegetables 103 outbreaks affecting 6,082 people ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

4. Introduction Standard plating Biosensor Highly specific/sensitive Highly specific/sensitive Time consuming Time consuming Expensive Expensive Sensitive and specific Sensitive and specific Rapid Rapid Simple Simple ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

5. Introduction Membrane Strip ImmunosensorMembrane Strip Immunosensor - Integrates antibody as a biological element and a conductive polymer element and a conductive polymer as an electrical transducer as an electrical transducer PolyanilinePolyaniline - Conductive organic material - Conductive organic material - High binding affinity with protein - High binding affinity with protein ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

6. Membrane Strip Immunosensor Schematic diagram of the biosensor ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

7. Immunosensor

8. Antigen Conjugate Antibody/Pani Antibody Water moleculeTheory Application Conjugate Capture region Absorption region region region

9. Methods & Materials Biosensor fabrication Sample preparation Detection process Confirmation ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

10. Biosensor preparation CelluloseApplication /Absorption padsCelluloseApplication /Absorption pads Nitrocellulose Capture padNitrocellulose Capture pad Fiber Glass membranes Conjugate padFiber Glass membranes Conjugate pad Conjugate pad preparationConjugate pad preparation Polyaniline was synthesized - Polyaniline was synthesized and conjugated with antibody and conjugated with antibody ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

11. Biosensor preparation Capture pad preparation Capture pad preparation -Purified antibody was immobilized on Nitrocellulose membrane. -Two electrodes (0.5 mm apart) were fabricated using a silver paste pen Construction Construction - Membranes were arranged and attached on a copper wafer - Sal, EHEC, EC biosensors were prepared - Sal, EHEC, EC biosensors were prepared ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

12. Sample preparation Produce samplesProduce samples - Iceberg lettuce, strawberries, and alfalfa sprouts and alfalfa sprouts InoculaInocula - Pure cultures of E. coli O157:H7, non- pathogenic E. coli, and S. Typhimurium - Mixture of the three organisms ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

13. Sample preparation Inoculation processInoculation process - 1ml of inoculum was added on the surface of the samples on the surface of the samples - Left for 45 min Cell recovery processCell recovery process - Samples were homogenized in 100ml of 0.1% peptone water (PW in 100ml of 0.1% peptone water (PW) ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

14. Sample preparation Filtration processFiltration process - Solution was filtered through a Bio-filter membrane a Bio-filter membrane - Membrane filter was vortexed in 10 ml of PW in 10 ml of PW - The concentrated solution - The concentrated solution was serially diluted was serially diluted ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

15. Detection Each biosensor was tested withEach biosensor was tested with 0.1ml of serially diluted sample Signal was measured atSignal was measured at 2, 4, and 6 min ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

16. Confirmation/Data analysis Standard platingStandard plating McConkey E. coli McConkey E. coli CHROM E. coli O157:H7 Brilliant Green S. Typhimurium Signal processingSignal processing - (Blank – inoculated samples) Statistical analysis using ANOVAStatistical analysis using ANOVA ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

17. Result Net resistance of EHEC biosensors used in lettuce sample inoculated with E. coli O157:H7

18. Result Net resistance of Sal biosensors used in sprouts sample inoculated with S. typhimurium

19. Result Specificity analysis on strawberries sample

20. Discussion Normalized signal Cell concentrations Response pattern Theory Data ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

21. Discussion Similar results were observed ( Kim 2000)Similar results were observed ( Kim 2000) Cell crowdingCell crowding - Saturates binding sites - Obstructs the charge transfer within the polymer structure within the polymer structure ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002

22. Conclusion The membrane strip immunosensor is: Highly specificHighly specific Highly sensitiveHighly sensitive - Detects as low as 10 CFU/ml RapidRapid - Detection completed in 6 minutes ASAE-CIGR CONGRESS ANNUAL INTERNATIONAL MEETING, 2002