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![Page 1: Predicting Cell Capture from Dilute Samples for ... cell capture-Mosier.pdf · Predicting Cell Capture from Dilute Samples for Microfluidic Biosensors Nathan S. Mosier and Bruce Craig](https://reader033.vdocuments.us/reader033/viewer/2022053015/5f1557f68a2a2332f97864e7/html5/thumbnails/1.jpg)
Predicting Cell Capture from Dilute Samples for Microfluidic Biosensors
Nathan S. Mosier and Bruce Craig
Department of Agricultural and Biological EngineeringLaboratory of Renewable Resources Engineering
Department of Statistics
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Acknowledgements› This research was supported through a cooperative agreement with
the ARS of the United States Department of Agriculture, project number 1935-42000-035.
› Eastern Regional Research Center
› Center for Food Safety Engineering
› Yifei Zang, Benjamin Tyner
› Laboratory of Renewable Engineering Faculty, Staff, and Students– Dr. Michael R. Ladisch, LORRE Director
– Dr. Tom Huang, Xingya Liu, Amanda Stewart, Wan-Tzu Chen, and Miroslav Sedlak
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Food Borne Pathogens
Feces/manure
Raw meat
Soil, food processing environment
Common sources
As few as 10 cellsunknownInfectious dose
Hemorrhagic colitis Gastroenteritis
Stillbirth
Death
Symptoms
2-5 days> 1-5 daysIncubation period
E. coli O157:H7L. monocytogenesBACTERIA
www.foodsafety.gov http://www.cfsan.fda.gov/~mow/intro.html
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Listeria monocytogenes
Gram positive (1x 2 μm )
Grows at 1 to 45 ºC
Acid and salt tolerant
Annual cases >2,500; Mortality 20-28%
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Rapid detection of bacteria
NoNoYesYes Live/Dead
YesYesNoYesIdentification
Growth Based
Nucleic Acid-based
Cell Comp. Based
Viability Based
Technique
Features
Growth steps required to provide live/ dead information
Using selective media
Our research addresses growth-based and cell-component based approaches to provide
live/dead and ID information
Through antibody-based capture
How/why?
Adapted from M.R. Ladisch, presentation to USDA-ARS-EERC, 8 November 2004
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2. Macro Level,
16 wells on a cartridge
1. Petri Dish
3. Micro Level,
On-Chip culture
Evolution of Growth Detection
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NeedsRapid detection during food processing
Elapsed time between sampling and result (time to result) of 3 hours or less
Detection against background of other non-pathogenic organisms
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Pathogen Detection on Biochips
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Microchips: Can be made in large numbers
•Microchips for adsorption studies
•PECVD fabricated oxide layer
•SiO2 with Pt patterns
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Jennifer Sturgis, BMS
Paul Robinson, BMS, BME
Arun Bhunia, Food Sci
Rashid Bashir, ECE
Rafael Gomez, ECE
Glass cover
In/Out ports
Channels/Wells
Epoxy adhesive
Pin
10 mm
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Silicon substrate
Pt electrode SiO2 insulation
Glass coverGlass cover
Z Z
Target molecules Target molecules bound to receptorsbound to receptors
Other molecules in theOther molecules in thesolution will solution will notnot bindbind
Receptors immobilized on electrode
Protein Biochip Technology Platform
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Micro-fabricated Biochip for Bacterial Cell Culture
PC boardw. heater
Wire-bond(with epoxy)
Micro-fluidicTubes
Micro-fluidicTubes
Edge Connector
MicroscopeObjective
BioChip
Measurementelectrodes
DEP captureelectrodes
Outlet Inlet
Measurementelectrodes
DEP captureelectrodes
Outlet Inlet
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Challenge
Food Sample
Detection volume
ERRC, 2000
10-100 mL
Dilute Cells
0.10-10 uL
Captured Cells
>1000x size change
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General strategy for present work:Recovery of live bacteria
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2. Extract Microorganisms
In order of decreasing severityblender > stomacher > massage
machine2 min air
(froth)20°CDecant intostomacher bag
2 minin air
20°CIn stomacher
bag
2 minin air20°CIn stomacher
bag
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Homogenized Hotdog ExperimentBefore blending the hotdog
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Homogenized Hotdog Experiment After hotdog was blended
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Massaged Hotdog Experiment
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General strategy for present work:Recovery of live bacteria
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25 mm filter holder25 mm filter holder
47 mm filter holder47 mm filter holder
Air SpaceAir Space
CCR Kit Assembly•Microfiltration basis
•Designed to capture and recover bacterial cells from a food-derived sample
•Uses membranes in series to process 100 mL hot dog extract into 0.1 to 1 mL sample
Cell Concentration and Recovery
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CCR Method
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CCR Work Summary› Careful selection of membranes
– Pore size and distribution
– Surface chemistry
– Interaction of target cell
2.5µm depth filter – 47mm diaPRE-FILTER
0.4µm screen filter – 25mm diacapture and conc. bacteria
Top downSide viewTop downSide view
1µm10µm
SEM by Chia-Ping Huang
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Depth Filter Before Use
WhatmanWhatman 42 42
10μ1800X 5kV41133
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Screen Filter Before Use
0.4 micron Polycarbonate0.4 micron Polycarbonate
1μ8000X 5kV41135
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Membrane Properties
Polycarbonate membrane
1 μm
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Bacterial cells recovered in 500 µLfrom hot dog meat broth containing 15 cells/ mL
y = 10.352x - 1.2721R2 = 0.9863
0
200
400
600
800
1000
1200
0 20 40 60 80 100Filtered volume(ml)
num
ber
of c
ells
rec
over
ed
cell number vs. filtered volume
Linear (cell number vs. filteredvolume)
Chen et al, 2004
~1500 cells/mL
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Validating Location of CellsNeed to “see” cells for rapidly screening different filtration and
sensing systems:
led to use of GFP bacteria
red or green E. coli
green L. monocytogenes
(Applegate and Sedlak, 2004)
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Wilson et al. (2001) Infect. Immun.
Mutant GFP protein – GFP-mut3
Fortineau et al. (2000) Res. Microbiol.
Mutant GFP protein – GFP-mut1Listeria monocytogenes ATCC 23074
Expressing Fluorescent Proteins
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GFP L. monocytogenes
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RFP E. coli and GFP L monocytogenes
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Sampling Issue
› How high a concentration is needed in sample introduced to biochip to guarantee detection of cells?
› How much volume must be processed from hot dog sample to guarantee capture of at least one cell?
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SYSTEM CHARACTERIZATION
› Uneven distribution of microbes – Graininess;
› Uneven distribution of sample matrices.
› Variability
› Poor reproducibility
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Distribution of GFP E. coli
108 cells/mL10X
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Distribution of GFP E. coli
107 cells/mL10X
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Distribution of GFP E. coli
106 cells/mL10X
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Distribution of GFP E. coli
105 cells/mL10X