Pathogen Capture and Concentration on Functionalized Polycarbonate - - PowerPoint PPT Presentation
Pathogen Capture and Concentration on Functionalized Polycarbonate Membrane: Detection and Sample Preparation Based on Immuno-filters Wan-Tzu Chen 1,2 , Tao Geng 3 , Arun K. Bhunia 3 , and Michael R. Ladisch 1,2,4 1 Department of Biomedical
ACS Annual Meeting 03-30-04
Wan-Tzu Chen1,2, Tao Geng3, Arun K. Bhunia3, and Michael R. Ladisch1,2,4
1Department of Biomedical Engineering 2Laboratory of Renewable Resources Engineering 3Department of Food Science 4Department of Agricultural and Biological Engineering
DEPARTMENT OF
Chia-Ping Huang and Dr. Debra Sherman
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
Gram-positive, rod-shaped bacterium
Highly resistant to salt and low temperature Cause listeriosis Average death rate of 20~ 30 % Especially harmful for pregnant women Occur in milk, cheese and ready-to-eat dairy
food via post-processing contamination
Conventional Sample Preparation Method*
Enrichment broth Oxford or LPM agars TSBYE agar
Commonly Used Detection
ELISA-High detection limit Immunobeads-Small volume PCR-False-positive results* * Surface Plasma Resonance
* Hitchins AD. 1998, Bacteriological Analytical Manual, AOAC International * * Manzano M et al. 1997, J. Sci. Food. Agric.,74: 25-30
Detect low level of foodborne pathogen
In complex and various foods In quick and precise way
Sample preparation usually is rate-limiting
But less attention is paid in this step
How do w e com bine tw o crucial steps
into one?
Antibodies Bacteria Membrane
Mixed Cellulose Polycarbonate PVDF Nylon
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 1 mL 5 mL 10 mL 25 mL 50 mL Filtered volumes (ml)
Concentration factors
Polycarbonate 0.4 um Mixed cellulose 0.45 um Expected concentrated factors
Antibody P66 or BSA
O
O
C CH 3 CH 3 C O N H (C H 2) 4 CH C O NH O C CH NH N(C H 2)4) CH (C H 2)3 CH N
Protein
O
O
C CH
3CH
3C O N H (CH
2) 4CH C O NH O C CH NH N(CH 2)4) CH (CH 2) 3 CHO
Proteins in PBS, pH 7.4
O O C C H
3C H
3C O N H (C H
2) 4C H C O N H O C C H N H
2(C H 2) 4N H
Glutaraldehyde in PBS, pH 7.4
O O C C H
3C H
3C O
Poly-L- Lysine, in Na2CO3
Polycarbonate
Suye et al., 1998, Biotechnol Appl
ACS Annual Meeting 03-30-04
2 4 -w ell Plate P6 6 Mem branes Add Bacterial Sam ples
LG surface + FITC-P66 Membrane+Poly-Lysine+Glutaraldehyde (LG surface)
50 100 150 200 250 300 Blank Blank+FITC-P66 Lysine- Glutaraldehyde+ FITC-BSA (Covalent binding) Lysine- Glutaraldehyde+ FITC-P66/BSA (Covalent binding) Fluorescence Intensity 1 2 3 4
Integration time = 8 ms
Initial= 3.7*108 cells Listeria on LG surface– HIGH non-specific binding! Listeria on BSA surface- blocking non-specific binding
Antibody BSA Blank LG surface Listeria on P66 surfaces Initial=3.7*108 cells
Initial= 3.7*108 cells
Blank LG surface
Initial= 3.7*108 cells Listeria on P66 surface E coli on P66 surface
ACS Annual Meeting 03-30-04
5 m l/ m in controlled by syringe pum p PBS containing L.m onocytogenes
P6 6 Mem brane
I nitial= 7 .3 x1 0 7 cells/ m l x 5 0 m l= 3 .7 x 1 0 9 cells 1 2 3 3 2 1 Blank Mem brane E coli on P6 6 Mem brane
500 1000 1500 2000 2500 Blank membrane E coli captured by P66 membrane
captured by P66 membrane Fluorescence Intensity
Integration time = 250 ms
Blank Mem brane E coli on P6 6 Mem brane
I nitial= 7 .3 x1 0 7 cells/ m l x 5 0 m l= 3 .7 x 1 0 9 cells
Hydrophilic spacer and cross-linker helps
antibody immobilization
Selective capture for batch incubation Mixture of two different bacteria can be
differentiated
Bacteria concentration and selective
capture can carry out simultaneously
Real-time concentration and detection can
be achieved within an hour