Development of Rapid
Through-put Nanotoxin Bioassays
Perry Kanury
Cassandra Viéville ~ Dr. Stacey Harper
CBEE/EMT
Background:
• Nanomaterials are widely used in both industrial
and consumer applications
• Currently thousands of distinct compounds in
use, with more introduced every day
o
Found in every day products such as deodorant, sunscreen,
Teflon, pesticides, etc.
• Little or no knowledge of the true environmental
or molecular impacts of these materials
o
Distinct lack of methods or protocols for toxicity testing
What are nanomaterials?
 Materials with at least one dimension between
1 and 100 nanometers
o Planar: Single dimension in ‘nano’ scale
 Example: Graphite sheet
o Tubular: Two dimensions in ‘nano’ scale
 Example: Bucky Tube
o Globular: Three dimensions in ‘nano’ scale
 Example: Bucky Ball
What are nanoparticles?
o Subcategory of nanomaterials
 Formerly known as 'Ultra Fine Particles'
 At least two dimensions between 1 and 100
nanometers in diameter, implying either tubular or
globular shapes
 Can take forms such as 'Nanoclusters', 'Nanocrystals',
or 'Nanopowders' depending on agglomeration behavior
 Wide range of applications including biomedical,
optical and electronic fields
 Often exhibit unique structure-property characteristics
as a result of volume to surface area ratio and
surface structure
 For reference: A typical nanoparticle has an effective
diameter of approximately 1/800th that of a human hair.
What does this mean to us?
 Wide range of possible effects to both human
and ecological systems
Examples:
 Introduction of antimicrobial agents such as
Aluminum Zirconium Tetrachlorohydrex (active
ingredient in deodorant) into a town’s microbe-based
water treatment system and subsequent watershed
 Potential endocrine disruption (among additional
myriad of effects) upon chronic exposure to
Titanium Dioxide (active ingredient in many
sunscreens) near lymph nodes, etc.
o Possible disruption of ‘Lock and Key’
hormone identification mechanisms within the
Endocrine system.
Need:
• R
apid testing strategies are necessary to identify specific
nanomaterials that result in toxicity in order to mitigate
risks from exposure and define structure-property
relationships that can be used to predict nanomaterial fate
and hazard in lieu of empirical data.
Hypothesis:
An aquatic ecosystem can be modeled in the form of a
‘nanocosm’ ~ an extremely small tritrophic ecosystem (250 uL).
Prediction:
This ecosystem can be effectively used for the purpose of
assessing the toxicity of nanoparticles in aquatic
environments, as well as modeling the impacts of these
nanoparticles on said ecosystems.
Another way of looking at it:
Another way of looking at it:
‘Nano’ Algae Ciliate Bacteria
(All 3 organisms)
Varying nanoparticle exposures
The makeup and utilization of a Nanocosm:
Tri-trophic aquatic ecosystem based upon our
local temperate region
 Photo-synthesizer (primary producer):
Chlamydomonas reinhardtii (green algae)
 Predator (primary consumer): Tetrahymena
thermophilia (ciliate)
 Decomposer (detritovore): E.coli (bacteria)
o Suspended in a defined microbial media
o Upon exposure by nanotoxins, can monitor
population dynamics and predator/prey
interactions to understand effects.
o
‘Nano’ Algae Ciliate Bacteria
(All 3 organisms)
Varying nanoparticle exposures
Typical Plate Layout
Exposure
Concentration
Nano
Bacteria
Algae
Ciliate
0.01 ppm
0.05 ppm
0.1 ppm
0.5 ppm
1 ppm
5 ppm
10 ppm
50 ppm
Three replicates of each treatment for each organism
How we observe these interactions:
‘Nano’ Algae Ciliate Bacteria
(All 3 organisms)
Flow Cytometer
Quantifies and
categorizes cells via
light diffraction and
fluorescence.
Varying nanoparticle exposures
Cell Concentration (cells/uL) of each
organism are obtained and analyzed based
upon effective size and fluorescence.
How we observe these interactions:
Tetrahymena Thermophilia AgNO3 Exposure over 5 days
How we observe these interactions:
Concentration Response
Treatment is observed relative to negative control within the same timepoint
(((treatment-control)/control)*100)
Future Work:
• Investigation of more ideal organisms than Tetrahymena
thermophilia in order to streamline data collection utilizing
existing equipment
• Investigation of additional endpoints beyond mortality
• Investigation of possible mechanisms of toxicity
• Establishment of a database of the effects of various
nanotoxin exposures.
Acknowledgements:
•
•
•
•
•
•
Dr. Stacey Harper
Cassandra Viéville
Howard Hughes Medical Institute
Environmental Health Sciences Center (EHSC)
Oregon State University
Dr. Kevin Ahern