Biofunctional membranes for Listeria monocytogenes detection - - PowerPoint PPT Presentation

Biofunctional membranes for Listeria monocytogenes detection

Wan-Tzu Chen 1,2, Michael R. Ladisch 1,2,4, Tao Geng 3, and Arun K.Bhunia 3

1 Department of Biomedical Engineering, Purdue

University

2 Laboratory of Renewable Resources Engineering,

Purdue University

3 Department of Food Science, Purdue University 4 Department of Agricultural and Biological

Engineering

DEPARTMENT OF

Acknowledgements

• Dr. Richard Linton
• Dr. Rashid Bashir
• Dr. Deb Sherman and Chia-Ping Huang
• Dr. Soo-Il Lee

This research was supported through a cooperative agreement with the ARS of the United States Department of Agriculture project number 1935-42000- 035

DEPARTMENT OF

Foodborne Pathogens

Bacteria, Virus and Parasite Potential Hazardous Food

Temperature, acidity, time, oxygen…..etc

Syndromes

Abdominal pains, vertigo, diarrhea, intoxication….etc

Examples

• C. botulinim, Salmonella, L. monocytogenes

Listeria monocytogenes

Gram-positive, rod-shaped bacterium

Highly resistant to salt and low temperature

Conventional Sample Preparation Method (BAM, 1998)

Enrichment broth Oxford or LPM agars TSBYE agar

Rapid Sample Preparation Method

Membrane filtration Decreased culture time

Syringe Filter System

PVDF Nylon Mixed cellulose Polycarbonate In 0.45 um and 0.22 um nominal size Inoculated hotdog meat broth (HDM) ~700 cells/ml Membrane filters Different volumes being filtered Membrane immersing in 0.5 ml of PBS with 1% of Tween 20 for 30 minutes Filtrates Bradford Protein Assay Plate out 100 µl on MOX agar

Tween 20

Non-ionic surfactant Produce surface liquid tension Prevent protein binding Hence help Listeria recovery

0.45 um with Tween 20

10 20 30 40 50 60 70 80 90 100 1 mL 5 mL 10 mL 25 mL 50 mL Filtered volumes (ml) Concentration factors

Polycarbonate 0.4 um Mixed cellulose 0.45 um Theoretic maximal concentration factors

Smaller Pore Sizes

10 20 30 40 50 60 70 80 90 100 1 mL 5 mL 10 mL 25 mL 50 mL Filtered volumes (ml) Concentrated factors Nucleopore 0.2 um Nucleopore 0.4 um MicronSep 0. 22 um MicronSep 0.45 um

Different Pore Structures

Mixed Cellulose (Depth filter) Polycarbonate (Screen filter) Listeria monocytogenes Ladisch, 2001

More Dilute Samples (15 cells/ml)

y = 10.352x - 1.2721 R

2 = 0.9863

• 100

100 300 500 700 900 1100 20 40 60 80 100 Filtered volume(ml) number of cells recovered

cell number vs. filtered volume Linear (cell number vs. filtered volume)

Volume below 0.13 ml results in no bacteria recovered Recovery rate~70% = 37 cells/ 20 ul

1800 cells/mL

New Thoughts

Membrane Antibodies Bacteria

Chemistry of Antibodies Immobilization

Antibody P66

Suye et al., 1998, Biotechnol Appl

• Biochem. 27: 245-248.

O O C C H 3 C H 3 C O C C H 3 C H 3 O O C O O C C H 3 C H 3 C O N H (C H 2) 4 C H C O NH O C C H NH NH 2(C H 2)4 O O C C H 3 C H 3 C O N H (C H 2)4 C H C O NH O C C H NH N (C H 2)4) C H (C H 2)3 C H O O O C C H 3 C H 3 C O N H (C H 2)4 C H C O NH O C C H NH N (C H 2)4) C H (C H 2)3 C H N Enzym e

Poly-lysine G lutaraldehyde Enzym e

Poly-Lysine Glutaraldehyde Enzyme

Antibody Immobilization

LG surface + FITC-P66 Membrane+Poly-Lysine+Glutaraldehyde (LG surface)

Listeria on Different Surfaces

5*107 cells/ml Listeria on LG surface– HIGH non-specific binding! Listeria on BSA surface- blocking non-specific binding

Specific Binding

Antibody BSA Blank LG surface Listeria on P66 surfaces Initial=5*107 cells/ml

Non-specific E coli Binding

Initial= 5*107 cells/ml

• E. Coli on P66 surfaces

Blank LG surface

Conclusions

Membrane filtration helps separation Tween 20 treatment facilitates Listeria recovery Recovery increases as pore sizes decrease Antibody immobilization works well on PC Bacteria differentiation can be carried out