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Fundamentals Nanotech Agr Mar 17 05 As Presented
Fundamentals Nanotech Agr Mar 17 05 As Presented
Fundamentals Nanotech Agr Mar 17 05 As Presented
of Agricultural and Biological Engineering LORRE Purdue University and 2Center for Strategic Studies National Defense University
229th ACS Meeting, San Diego, CA, Paper 205, March 17, 2005
Acknowledgements
Research supported by USDA ARS Contract 1935-42000-035 National Defense University (DAB J29-03-P-0022) Richard Linton, Director, Center of Food Safety Engineering Randy Woodson Dean, School of Agriculture
DNA as a Barcode
Short specific DNA sequences for identifying species (cost of readers is decreasing) Cytochrome C oxidase I all species have the enzyme with variations from one species to another May work for birds but not plants need to identify another gene.
Marshall, Science, 2005
Microfluidics
Movement of fluids at microscopic level Micron-sized channels and features
Applications
Biosensors Micro-bioseparations Pathogen detection
PDMS
PDMS
The Need Simple and rapid fabrication Well defined surface chemistries Flexibility and adaptability Amendable for rapid prototyping techniques
SiO2 substrate
(a) (b)
Glass substrate
(c)
PDMS
t = 3 min
Flow channel
1 nL/mm channel
1 nL well at intersection
(b) t= ~3 minutes
Micro-scale Separation
Derivatize channels with ion exchange bioreceptor Assemble micro-device Use pre-derivatized particles
Surface
Hydroxyl Hydroxyl Carboxyl Sulfonate Amino Dimethylamino
Chemistry Size Surface (m) charge 0.1-7.9 -OH -OH -COOH -SO3 -NH3 -NH(CH3)2
0.7-7.9 0.7-7.9 0.7-7.9 0.7-7.9 0.7-7.9
---+ ++
Surface
Antibody (IgG) Avdin Streptavidin Biotin Protein A Protein G
pI
NA 10.0 5.0 NA 4.9 5.0
EtOH Ultra-sonic cleaning in EtOH for 5 min Dip-coat in protein or particle slurry Glass fiber coated with particles
+
Bare glass fiber, 12 m dia
Biotin
+
Streptavidin Glass fiber pre-coated with biotin-BSA
Micro-scale Separation
Derivatize channels with ion exchange or bioreceptor (in bead form) Assemble micro-device Carry out separation in flowing fluid Sample size = 1 L Apply sample at 0.25 to 1 nL / sec
Labeled avidin (green; 10 g/ml) and BSA (red; 10 g/ml) liquid mixture; t=0
PDMS
Glass substrate
Glass substrate
(a)
Microscope objective
HV power
Microchip
Oscilloscope
ANTIBODY
Antigen binding sites
Fab Fab N N S S S Fab S S S S S S Fc Fc S S S S S S S S S S S N S Fab S N
Disulfide bond
S S
Antigen
Specific Binding
BSA Antibody
Initial=3.7*108 cells
Blank LG surface
Listeria on P66 Ab
Blank LG surface
Fluorescence Intensity
2500
Fluorescence Intensity
2000
1500
1000
500
0 Blank membrane E coli captured by P66 membrane L. monocytogenes captured by P66 membrane
SEM Images
Initial= 7.3x107 cells/ml x 50 ml=3.7 x 109 cells
Blank Membrane
Tip of a pin
Channels/Wells
Epoxy adhesive
Microchips for adsorption studies PECVD fabricated oxide layer SiO2 with Pt patterns
To use chip
1. Sample fluid 2. Place fluid onto chip for interrogation 3. Electrically detect if receptor binds something present in the fluid Complete steps 1 through 3 in three hours. That something could be a pathogen that requires a rapid, preventative response.
Listeria monocytogenes
Food-born pathogen Gram positive (1m x 3 m ) Growth temperature (1-45 C) Acid and salt tolerant Cause listeriosis (fever, headache, meningitis, encephalitis, liver abscess, abortion & stillbirth) Annual cases >2,500; Mortality 20-28%
Bhunia, Jarat, 2001
Design principles
Microfabricate chip. Then Prepare surface so that nothing adsorbs (biochemical equivalent of Teflon) Fix protein receptor onto treated surface. Use nanogram amounts of receptors, since these proteins cost $10,000 per gram ( but only pennies per 1000 nanograms)
Combinations of Technology
DNA Barcode + RFID Tag + Microfluidic Sensor = Information What is it? Where is it? Where has it been? These capabilities have existed previously. Nanotechnology may make them affordable.
Applications in Foods
Food Products $13.1 billion in soda pop 7.5 billion in breakfast cereals 60.0 billion in fresh meats Branded meats, precooked products, micro-waveable red meats Livestock, poultry
Sales figures from Kilman, Wall Street Journal, Feb 20, 2002
Biodegradable sensors
Tracking foods during processing
Conclusions
Nanotechnology combined with biotechnology enables new sensors that are difficult to make otherwise Nanotechnology enables detection at a microscale: small, localized and rapid Applications for food and agriculture in identification of species,pathogens