A Membrane Strip Immunosensor for Foodborne Pathogen Detection

1
A Membrane Strip Immunosensor for Foodborne
Pathogen Detection

• Zarini Muhammad-Tahir
• Evangelyn C. Alocilja
• Michigan State University

2
Outline

• Introduction
• Methods and materials
• Results
• Discussion
• Conclusion

3
Introduction

• 90 of foodborne illnesses caused by bacteria
• Escherichia coli O157H7 and Salmonella
  typhimurium
• Illnesses mild, watery diarrhea,
• to life-threatening conditions
• Fatal children, elderly, and
• immune-compromised individuals
• Outbreaks related to fresh fruits and vegetables
• 103 outbreaks affecting 6,082 people

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Introduction

• Standard plating Biosensor

• Highly specific/sensitive
• Time consuming
• Expensive

• Sensitive and specific
• Rapid
• Simple

5
Introduction

• Membrane Strip Immunosensor
• - Integrates antibody as a biological
• element and a conductive polymer
• as an electrical transducer
• Polyaniline
• - Conductive organic material
• - High binding affinity with protein

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Membrane Strip Immunosensor
Schematic diagram of the biosensor
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Immunosensor
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Theory
Application Conjugate Capture region
Absorption
region region region
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Methods Materials
Biosensor fabrication
Sample preparation
Detection process
Confirmation
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Biosensor preparation

• Cellulose Application /Absorption pads
• Nitrocellulose Capture pad
• Fiber Glass membranes Conjugate pad
• Conjugate pad preparation
• - Polyaniline was synthesized
• and conjugated with antibody

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Biosensor 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
• - Membranes were arranged and attached on a
  copper wafer
• - Sal, EHEC, EC biosensors were
  prepared

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Sample preparation

• Produce samples
• - Iceberg lettuce, strawberries,
• and alfalfa sprouts
• Inocula
• - Pure cultures of E. coli O157H7, non-
  pathogenic E. coli, and S. Typhimurium
• - Mixture of the three organisms

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Sample preparation

• Inoculation process
• - 1ml of inoculum was added
• on the surface of the samples
• - Left for 45 min
• Cell recovery process
• - Samples were homogenized
• in 100ml of 0.1 peptone water (PW)

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Sample preparation

• Filtration process
• - Solution was filtered through
• a Bio-filter membrane
• - Membrane filter was vortexed
• in 10 ml of PW
• - The concentrated solution
• was serially diluted

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Detection

• Each biosensor was tested with
• 0.1ml of serially diluted sample
• Signal was measured at
• 2, 4, and 6 min

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Confirmation/Data analysis

• Standard plating
• McConkey E. coli
• CHROM E. coli O157H7
• Brilliant Green S. Typhimurium
• Signal processing
• - (Blank inoculated samples)
• Statistical analysis using ANOVA

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Result
Net resistance of EHEC biosensors used in lettuce
sample inoculated with E. coli O157H7
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Result
Net resistance of Sal biosensors used in sprouts
sample inoculated with S. typhimurium
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Result
Specificity analysis on strawberries sample
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Discussion

• Response pattern

Normalized signal
Cell concentrations
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Discussion

• Similar results were observed ( Kim 2000)
• Cell crowding
• - Saturates binding sites
• - Obstructs the charge transfer
• within the polymer structure

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Conclusion

• The membrane strip immunosensor is
• Highly specific
• Highly sensitive
• - Detects as low as 10 CFU/ml
• Rapid
• - Detection completed in 6 minutes