fundamentals nanotech agr mar 17 05 as presented

8/2/2019 Fundamentals Nanotech Agr Mar 17 05 as Presented

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Fundamentals of Nanotechnologyfor Agriculture

Michael Ladisch1, Tom Huang1, Nathan Mosier1, andRobert Armstrong2

1

Department of Agricultural and Biological EngineeringLORRE

Purdue University

and2Center for Strategic Studies

National Defense University

229th ACS Meeting, San Diego, CA, Paper 205, March 17, 2005


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Acknowledgements

Research supported by

USDA ARS Contract 1935-42000-035National Defense University(DAB J29-03-P-0022)

Richard Linton,Director, Center of Food Safety Engineering

Randy WoodsonDean, School of Agriculture


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Nanoscience and Nanotechnology

Nanoscience:Fabrication, study and modeling of devicesand structures where at least one dimension

is 200 nm or smaller.Nanotechnology:

Enables devices that are compact, portable,energy efficient, integrate sensing, and carryout complex functions of a full-scale laboratory


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DNA as a Barcode

Short specific DNA sequences for identifyingspecies (cost of readers is decreasing)

Cytochrome C oxidase I

all species have the enzyme with variationsfrom one species to another

May work for birds but not plants need toidentify another gene.

Marshall, Science, 2005


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Radio Frequency Identification as a

BarcodeRFID (Radio Frequency Identification)

Integrated circuit on a small silicon chip attachedto a small, flexible antenna, creating a tag.Integrated circuit provides data storage to record

and store information.Reader sends a signal to the tag.

tag absorbs some of the RF energy from thereader signal and then reflects the RF energy asa return radio signal containing information fromits memory.

From Alien website: http://www.alientechnology.com


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Microfluidics

Movement of fluids at microscopic levelMicron-sized channels and features

ApplicationsBiosensorsMicro-bioseparations

Pathogen detection


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Benefits: Microfluidic Systems-Interrogation of fluid samples

Miniaturization

Consumes less reagentsEnables higher sensitivity

Shorter analysis time


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Rapid Prototyping using PDMSUV

High resolution transparency as maskphotoresist

SiO2 or silicone substrate

Develop photoresist obtains master

Pour PDMS over master

Cure 70 C for 1 hour

PDMS

PDMS Release PDMS from master and

seal against a flat substrate

Microchannels formed.PDMS

Process takes a day or more


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The Need

Simple and rapid fabrication

Well defined surface chemistries

Flexibility and adaptability

Amendable for rapid prototypingtechniques


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Microfiber Assisted Fabrication in 15 min

Flat PDMS cover

Glass fiber (~12 m diameter)

PDMS

SiO2

SiO2, glass, or

PDMS substrate

1 L labeled avidin (green)

Glass fiber

t = 3 min

Glass substrateSiO2 substrate

(a) (b) (c)

Flow

channel

1 nL/mm channel


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Mixing in Nanoliter Well

Glass fiber (~12 m dia)

PDMS

(a)SiO2 substrate

1 nL well at intersectionLabeled BSA

(red, 200 g/ml)

Labeled avidin

(green, 200 g/ml) t= ~3 minutes(b)


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Micro-scale Separation

Derivatize channels withion exchange

bioreceptor

Assemble micro-device

Use pre-derivatized particles


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Commercially Available Micro/Nano

ParticlesType Surface Chemistry Size

(m)

Surfacecharge

Silica Hydroxyl -OH 0.1-7.9 -

Polystyrene Hydroxyl -OH 0.7-7.9 -

Polystyrene Carboxyl -COOH 0.7-7.9 --

Polystyrene Sulfonate -SO30.7-7.9 ---

Polystyrene Amino -NH30.7-7.9 +

Polystyrene Dimethylamino -NH(CH3)20.7-7.9 ++

Data from Spherotec, Inc. Libertyville IL.


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Protein Coated Micro/Nano Particles

Type Surface pI Size

(m)

SurfaceAffinity

Polystyrene Antibody (IgG) NA 0.7-7.9 Bindsprotein A, G

Polystyrene Avdin 10.0 0.7-7.9 Binds tobiotin

Polystyrene Streptavidin 5.0 0.7-7.9 Binds tobitoin

Polystyrene Biotin NA 0.7-7.9 Binds tostrept/avidin

Polystyrene Protein A 4.9 0.7-7.9 Binds to IgGantibody

Polystyrene Protein G 5.0 0.7-7.9 Binds to IgG

antibody

Data from Spherotec, Inc. Libertyville IL.


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Dip-coating Microfibers with Protein or

Derivatized ParticlesGlassfiber

EtOH

Ultra-soniccleaning inEtOH for 5 min

Dip-coat inprotein orparticle slurry

Glass fibercoated withparticles


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Glass Fiber Coated with DimethylaminoBeads

Dimethyl-aminomicrobeads, 800 nm

OH OH OH

+N CH33

H

CH Bare glass fiber, 12 m dia


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Glass Fiber Coated with Streptavidin Beads

Streptavidinmicrobeads, 800 nm Biotin

+Glass fiber pre-coated

with biotin-BSAStreptavidin


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Micro-scale Separation

Derivatize channels with

ion exchange orbioreceptor (in bead form)

Assemble micro-deviceCarry out separation in flowing fluid

Sample size = 1 L

Apply sample at 0.25 to 1 nL / sec


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Microfluidic Separation Device

Labeled avidin (green; 10 g/ml) and

BSA (red; 10 g/ml) liquid mixture; t=0

Labeled avidin (green; 10 g/ml) and

BSA (red; 10 g/ml) liquid mixture; t=0

Glass fiber

coated with

dimethylamino

microbeads

Glass fiber

coated with

biotinylated

BSA

Glass substrate Glass substrate

PDMSPDMS

t= ~3 minutes t= 5 minutes(a) (b)

Huang et al, 2003


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Surface Effect in Microfluidic Channels

(particle detection or capture)

Hydrophobic vs. Hydrophilic

Surface chemistry of a microchannel is extremelyimportant in the movement and control of fluids atmicroscopic level

Water in hydrophobic channel Water in hydrophilic channel
http://microfluids.engin.brown.edu/images/HydroPhillic_Surface.jpg


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Detection of GFP E. coli in moving fluid

across microfiber device

1-2 m GFP E. coli

(~106

cells/ml) in PBS

1-2 m GFP E. coli

(~106

cells/ml) in PBS

linear velocity of 10 um/s Linear velocity of 100 um /s


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Counting Cells Using Micro-device

PMT

HV power

Air slit

Microscopeobjective

Microchip Oscilloscope

ANTIBODY


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ANTIBODYANTIBODY

Fc

Fab Fab

SS

SS

SS

SS

SS

SSS

S

SS

SS

SS

SS

SS

N NNN

Fc

Fab Fab

Antigenbinding

sites

Disulfide

bond

Antigen

Surface of a biochip

* 3-D IgG molecule is obtained from David Wild (http://www.techfak.uni-bielefeld.de/bcd/ForAll/Introd/antibody.html).

Antibody

antigen

interaction
http://www.techfak.uni-bielefeld.de/bcd/ForAll/Introd/antibody.htmlhttp://www.techfak.uni-bielefeld.de/bcd/ForAll/Introd/antibody.html


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Specific Binding

Antibody

BSA

Blank LG surface Listeriaon P66 Ab

Initial=3.7*108 cells


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E coliDoes Not Bind

Initial= 3.7*108 cells

E. Colion P66 surfacesBlank LG surface


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Mixture of Two Bacteria

Initial= 3.7*108 cells

Listeriaon P66

surface

E colion P66

surface


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Flow-through: Syringe + Holder

5 ml/mincontrolled bysyringe pump

PBS containingL.monocytogenes

or E.coli

P66

Membrane


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Fluorescence Intensity

0

500

1000

1500

2000

2500

Blank membrane E coli captured by P66

membrane

L. monocytogenes

captured by P66

membrane

Fluorescen

ce

Intensity

Integration time = 250 ms


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SEM Images

BlankMembrane

E colion P66Membrane

L. monocytogenes

on P66 Membrane

Initial= 7.3x107 cells/ml x 50 ml=3.7 x 109 cells

Cl f P t i Bi hi


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Close-up of a Protein Biochip

Glass cover

In/Out ports

Channels/Wells

Epoxy adhesive

Pin

Tip of a pin

Well = 5 nanoliters30 to 50 wells/chip

Cross-section of a well on a Biochip


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Cross-section of a well on a Biochip

Silicon substrate

Pt electrode SiO2 insulation

Glass coverGlass coverTarget moleculesTarget molecules

bound to receptorsbound to receptors

Receptors immobilized

on electrode

Protein receptorProtein receptor

20 nm size20 nm size

Microchips can be produced in


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Microchips can be produced in

Large Numbers

Microchips for adsorption studies

PECVD fabricated oxide layer

SiO2 with Pt patterns


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To use chip

1. Sample fluid

2. Place fluid onto chip forinterrogation

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 thatrequires a rapid, preventative response.

Li t i t


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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%

Bhuni a, Jar at , 2001


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Sample From Hotdog

OilFat

Aqueous Sample

Solids


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Rapid Cell Concentration andRecovery Needed

Separate cells from food or agricultural

sample

Concentrate bacteria

Recover bacteria and introduce onto biochip


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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)


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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.


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Applications in FoodsFood 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 fromSales figures from KilmanKilman, Wall Street Journal, Feb 20, 2002, Wall Street Journal, Feb 20, 2002


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Detect Gene Expression Products

Animal health monitoringEarly warning of infectious disease

Biodegradable sensorsTracking foods during processing

Identity tracking and preservation

Nanoscale Science and Engineering

for Agriculture and Food Systems,CSREES, USDA National Planning Workshop, 2002


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Conclusions

Nanotechnology combined with

biotechnology enables new sensorsthat are difficult to make otherwise

Nanotechnology enables detection at amicroscale: small, localized and rapid

Applications for food and agriculture inidentification of species,pathogens


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fundamentals nanotech agr mar 17 05 as presented

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