msel mel cstl metrology for the “fate” of nanoparticles in biosystems michael t. postek, andras...
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Metrology for the “Fate” ofNanoparticles in Biosystems
Michael T. Postek,Michael T. Postek,Andras Vladar, Thomas LeBrun and John Dagata, MELAndras Vladar, Thomas LeBrun and John Dagata, MEL
John Small, John Small, John Henry Scott, Scott Wight, Peter Barker, CSTL John Henry Scott, Scott Wight, Peter Barker, CSTL
Alamgir Karim, Alamgir Karim, Jack Douglas, Matthew Becker, Vincent Hackley, Jack Douglas, Matthew Becker, Vincent Hackley,
Andrew Allen, Stephanie Hooker, MSELAndrew Allen, Stephanie Hooker, MSEL
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Michael T. Postek, ChampionAndras VladarThomas LeBrunJohn Dagata,
Michael T. Postek, ChampionAndras VladarThomas LeBrunJohn Dagata,
John Small, ChampionJohn Henry ScottScott Wight, Peter Barker
John Small, ChampionJohn Henry ScottScott Wight, Peter Barker
Alamgir Karim, Champion Jack DouglasMatthew BeckerVincent Hackley Andrew AllenStephanie Hooker
Alamgir Karim, Champion Jack DouglasMatthew BeckerVincent Hackley Andrew AllenStephanie Hooker
Metrology for the “Fate” of Nanoparticles
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Metrology for the “Fate” of Nanoparticles
• Competence proposal is motivated by work stemming from:Competence proposal is motivated by work stemming from:– National Nanotechnology InitiativeNational Nanotechnology Initiative
• Subcommittee on Nanoscale Science, Engineering and Technology (NSET)Subcommittee on Nanoscale Science, Engineering and Technology (NSET)– Michael PostekMichael Postek– Alamgir KarimAlamgir Karim
• Nanotechnology Environmental and Health Implications Working Group (NEHI WG)Nanotechnology Environmental and Health Implications Working Group (NEHI WG)– John SmallJohn Small– Michael PostekMichael Postek
– The National Institute for Occupational Safety and Health (NIOSH)The National Institute for Occupational Safety and Health (NIOSH)• Andrew Maynard (endorsement)Andrew Maynard (endorsement)
– Environmental Protection Agency (EPA)Environmental Protection Agency (EPA)• Barbara KarnBarbara Karn• Nora SavageNora Savage
…. “I would anticipate a highly successful project, and one that will support research into the impact of nanotechnology in a significant manner.” ……. Andrew Maynard, NIOSH
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NIST is the only organization that has the mission and the necessary combination of staff expertise, experience, industry contacts, industry respect, & worldwide recognitionstaff expertise, experience, industry contacts, industry respect, & worldwide recognitionas the leading measurement and standards institution to address the high risk scientific and technological challenges
associated with developing the measurement techniques and technical standards needed in this new field.National Nanotechnology Initiative, pg 119.
Why Now…Impact on Society, Environment, and Commerce
Without NIST’s contributions…..the lack of a common infrastructure for bioscience measurements, standards, & data willthe lack of a common infrastructure for bioscience measurements, standards, & data will
constrain innovation and stifle the commercialization of new bioscience-based applicationsin health care, agriculture and food, environmental protection, homeland security, and many other areas.
NIST 2010 Strategic Plan, pg 11.
…A joint collaboration with the National Institute of Standards and Technology (NIST) and the FDA is a high priority. The collaboration will focus on developing standards The collaboration will focus on developing standards for nanoscale devicesfor nanoscale devices
and both in vitro and in vivo characterization assays that could serve as a starting point for regulatory filings.Cancer Nanotechnology Plan, pg 24.
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Materials TodayJuly/Aug 2003: 64
Why Now...Potential for Catastrophic Effects: Psychological and Economic
X Flawed Science Big PRESS
For Science, Nanotech Poses Big Unknowns
By Rick WeissWashington Post Staff WriterSunday, February 1, 2004; Page A01
This approach will not reassure the public at largeNANOTECHNOLOGY RESEARCH GRANTS INVESTIGATING ENVIRONMENTAL
AND HUMAN HEALTH EFFECTS OF MANUFACTURED NANOMATERIALS: A
JOINT RESEARCH SOLICITATION-EPA, NSF, NIOSH
• Basically a toxicology study of nanoparticles
• Need for known characterized particles
• Need for standardized methodology
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VA
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S&T DEVELOPMENT OVER TIME
NewInvention
Period
ScientificDiscovery
March 2004
A new product development toolkit
containing powerful new scientific and
technical methods ……. is urgently needed to improve predictability and efficiency along the critical path from laboratory concept to
commercial product.
*Office of Cellular, Tissue, and Gene Therapies
Why NIST…Lack of Measurement Infrastructure has Become a Critical Hurdle to Further Innovation
StandardsDevelopment
(Phase II)
MeasurementMethods & Tools
(Phase I)
BasicScience
NIST’sRole
TechnologyImprovement
Period
“Instrumentation and metrology crosscut all the NNI Grand Challenges, and are vital to the success of the NNI. Advances in fundamental nanoscience, design of new nano-materials, and ultimately manufacturing of new nanoscale products will all depend to some degree on the capability to accurately and reproducibly measure properties and performance characteristics at the nanoscale.
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It is this simple - what is the difference?
Left
RightRightNanometer sized particles of Zinc Oxide
Micrometer sized particles of Zinc Oxide
Primary Particle size of 3-10 nm
What is the effect on the body?
FDA approval of zinc oxide nanoparticles is “grandfathered” in because of the previous approvals for micrometer-sized particles…… discussion held at the first NEHI meeting
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Competence Goal
• What we want to do:– Develop an innovative toolbox for metrology of nanoparticles in biological systems that currently does
not exist• Focused Ion Beam sectioning• Cryo TEM/SEM/FIB stage integration• Cryomicrotomy• …..
– Form a strong group of experts in biological nanoparticle analysis• Develop an unbiased source of expertise
– Provide better technical support for other agencies
• What we are not doing:– Attempting to become NIH– Losing our metrology focus
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Motivation
Why Nanoparticle Metrology for Why Nanoparticle Metrology for Biological Systems?Biological Systems?
• Nanoparticles are being produced and are a part Nanoparticles are being produced and are a part of our environmentof our environment
• Nanoparticle measurements are challengingNanoparticle measurements are challenging• Biological samples must be carefully prepared Biological samples must be carefully prepared • Validation procedures are needed for Validation procedures are needed for
standardization of measurementsstandardization of measurements• NIST needs to develop competence in this areaNIST needs to develop competence in this area• NIST can make a differenceNIST can make a difference
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• Measurement ChallengeMeasurement Challenge• Technical StrategyTechnical Strategy• Issues and BarriersIssues and Barriers• Dissemination of ResultsDissemination of Results• MilestonesMilestones• Resources and LeveragesResources and Leverages
Outline
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• Measurement ChallengeMeasurement Challenge• Technical StrategyTechnical Strategy• Issues and BarriersIssues and Barriers• Dissemination of ResultsDissemination of Results• MilestonesMilestones• Resources and LeveragesResources and Leverages
Outline
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Underlying Basis for Measurement NeedUnderlying Basis for Measurement Need
Exposure/UptakeExposure/Uptake
BioactivityBioactivityTransportTransport
Nano-particleLife-Cycle
Depending upon the system, increased particle size can have either negative or positive consequences…….Particle size therefore can be indicator of expected activity.…. Mattison et al., 2001
“….An essential aspect of engineered nanomaterials is how their properties depend on physical and chemical structure. Quantitative assessments of the risk associated with engineered nanomaterials can not therefore proceed without techniques capable of providing biologically-relevant physiochemical information with nanometer resolution.” – Andrew Maynard, NIOSH
Effect of size
Control of mobility and distribution Surface chemistry effects
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Challenge: Biological Sample PreparationChallenge: Biological Sample Preparation
• Biological samples require:Biological samples require:– FixationFixation– DehydrationDehydration– EmbeddingEmbedding– SectioningSectioning
• Dual beam FIBDual beam FIB• CryomicroscopyCryomicroscopy• CryomicrotomyCryomicrotomy
• Provides a new paradigm for NISTProvides a new paradigm for NIST– Physical metrologyPhysical metrology
• Biological sample preparation is not new to NIST, but not extensively Biological sample preparation is not new to NIST, but not extensively practiced at NISTpracticed at NIST
• Biological samples require expertise that must be developed at NIST Biological samples require expertise that must be developed at NIST in order to assist our customers in metrology related situations.in order to assist our customers in metrology related situations.
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SWNT
DWNT
MWNT
Nanotubes (C, Au, Hybrid…)Quantum DotsDendrimers
Challenge: Diversity of Nanoparticles Presents Numerous Characterization Challenges
These materials will be the first “in the pipeline” as new products but,
new self-assembled soft material constructs with increasing difficult characterization challenges must be addressed
MicellesLiposomesFunctionalizedNanoparticles
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core diameter
shell thickness
Molecular mass& distribution
Aggregation number(# molecules per particle)
Core composition&
stiffness
Surface chemistry&
Extent of bio-functionality
Properties Impacted by Measurable ParameterspH susceptibilityBioactivityDiffusion coefficientAnisotropic transportMembrane permeabilityDegradation mechanisms and byproducts
Dispersion
Challenge: Numerous Measurement Parameters of Nanoparticles
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Challenge: Increasing Complexity
CC Lee, M Yoshida, JMJ Frechet, EE Dy, and FC SzokaBioconjugate Chem., 16(3), 535 -541, 2005
AW Jensen, BS Maru, X Zhang, DK Mohanty, BD Fahlman, DR Swanson, and DA TomaliaNano Letters, 5(6), 1171 -1173, 2005
C Loo, A Lowery, N Halas, J West, and R DrezekNano Letters, 5 (4), 709 -711, 2005
Dendrimer decoratedDendrimer decoratedPolymer BrushPolymer Brush
Fullerene decoratedFullerene decoratedDendrimerDendrimer
Antibody labeledAntibody labeledGold NanoshellGold Nanoshell
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Challenge: High Measurement Precision of Surface Chemistry
CM Sayes, JD Fortner, W Guo, D Lyon, AM Boyd, KD Ausman, YJ Tao, B Sitharaman, LJ Wilson, JB Hughes, JL West, & VL Colvin“The Differential Cytotoxicity of Water-Soluble Fullerenes” Nano Letters, 4 (10), 1881 -1887, 2004
Variable surface chemistry induces6 orders of magnitude6 orders of magnitude
difference in toxicity profile
Small Changes Cause Enormous Differences in the Bioactivity
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Challenge: Multiple mechanisms of particle translocation
Computational approaches can help elucidate potential mechanisms of translocation
Modeling is Imperative
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• Measurement ChallengeMeasurement Challenge• Technical StrategyTechnical Strategy• Issues and BarriersIssues and Barriers• Dissemination of ResultsDissemination of Results• MilestonesMilestones• Resources and LeveragesResources and Leverages
Outline
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Strategic Technical Approach
BareNanoparticle
Characterization
Cell Culture
Sample Preparation
Flocculation Particle Tracking
Extra & Intra Cellular Nanoparticle Characterization
Transport Properties
Simulations Modeling
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A Multi-laboratory EffortA Multi-laboratory Effort
Modeling
Cell Culture Specimen Preparation
Microscopy
Analytical
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Initial Test Nanoparticles and Metrology
Gold nanoshell
Extracellular Metrology Intracellular Metrology
Cell Culture Ultrastructure
Particle Characterization
TEM TEM (sectioned material)
SEM SEM (sectioned material)
FIB (sectioned material)
Cryomicroscopy
Dimensional Metrology SEM SEM
TEM TEM
DLS
USAXS
Particle Imaging SEM
TEM
Fluorescence Fluorescence Fluorescence
AFM AFM AFM
Transport Modeling x x x
Particle Placement Optical Tweezers Optical Tweezers
Particle Localization Fluorescence Fluorescence
Particle Removal Optical Tweezers
Quantum Dot
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A Multi-laboratory Effort
Modeling
Cell Culture Specimen Preparation
Microscopy
Analytical
Nanometrology
Single Particle Analysisand Manipulation
Transmission Electron Microscopy• NEW Aberration Corrected TEM
• Potential New NIST competenceScanning Electron Microscopy• NEW Environmental SEM
•Potential New NIST competenceOptical Tweezers• Single nanoparticle manipulation in biological materials
•Potential New NIST competenceAtomic Force Microscopy of biological materials
3-D reconstruction of cells and particles
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A Multi-laboratory Effort
Modeling
Cell Culture Specimen Preparation
Microscopy
Analytical
Nanometrology
Single Particle Analysisand Manipulation
Transmission Electron Microscopy• NEW Aberration Corrected TEM
• Potential New NIST competenceScanning Electron Microscopy• NEW Environmental SEM
•Potential New NIST competenceOptical Tweezers• Single nanoparticle manipulation in biological materials
•Potential New NIST competenceAtomic Force Microscopy of biological materials
3-D reconstruction of cells and particles
STEM EDS maps with 50 nm resolution at 5mm WD
C Fe
Overlay
SiK
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A Multi-laboratory Effort
Modeling
Cell Culture Specimen Preparation
Microscopy
Analytical
Nanometrology
Focused Ion Beam• NOVA NanolabBiological Sample Preparation• UMd
NOVA Nanolab - dynamicsectioning of biologicalmaterial fornanoparticle analysisand metrology• Potential New NIST competence
Biological sample preparation for electron microscopy• Potential New NIST competence
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A Multi-laboratory Effort
Modeling
Cell Culture Specimen Preparation
Microscopy
Analytical
Nanometrology
X-ray Scattering
FLOW-CELL SCHEMATIC
Ultra-Small Angle X-ray Scattering (USAXS)
Advantages of USAXS facility• Probes dimensions from nm to µm scale with good statistics• Small (adjustable) beam size (0.2 x 2 mm2) permits µL samples• Absolute scattering cross-section permits quantitative analysis• X-ray energy (wavelength) tunability• High beam flux at sample (>1012 photons/s at 10 keV)
APS
33IDundulator
Si (111)monochromator
mirrors
2D slits
Si (111)collimating
crystals
ionchamber
sample
Si (111)analyzercrystals
photodiodedetector
IoI
E
USAXS SLIT-SMEARED GEOMETRY
USAXS compliments the microscopy methods, providing statistically relevant data on populations of particles under in situ conditions
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A Multi-laboratory Effort
Modeling
Cell Culture Specimen Preparation
Microscopy
Analytical
Nanometrology
Dynamic Light Scattering
Dh = Hydrodynamic diameter of SCK
Dynamic Light Scattering (DLS)
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• Measurement ChallengeMeasurement Challenge• Technical StrategyTechnical Strategy• Issues and BarriersIssues and Barriers• Dissemination of ResultsDissemination of Results• MilestonesMilestones• Resources and LeveragesResources and Leverages
Outline
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Issues/Barriers
• Dynamic Light Scattering (DLS) of particles results in the hydrodynamic diameter which is highly influenced by particle shape and solvation
• Sample preparation for complimentary electron beam techniques generally requires drying and good dispersal
• Images are highly influenced by electron beam interaction and charging effects
• Comparisons of DLS measurements with SEM, TEM and other techniques must be done
– But will result in different values.
• Resolution of these measurement differences is imperative
– Important part of this competence effort
5 nm size Pt particles in their original environment (fuel cell)
Microscopy Measurements
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1) Endocytosis and the Encapsulation of Nanoparticles by Clathrin
2) Computational of Transport and Configurational Properties of
Nanoparticles
3) Influence of Particle-Substrate Interactions on the Rate of Diffusion
4) Matrix-Driven Translocation of Nanoparticles
Numerous proposed mechanisms and controversy in the scientific literature motivates intensive experimental and computational study of these types of assembly processes.
Modeling
Nanoparticle are unique in that they are commensurate in size with proteins & other biopolymers. This simple fact accounts
for their high degree of bio-activity.
There is currently no systematic experimental or computational approach to predict how nanoparticles
might affect normal biological processes.
Issues/Barriers
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cell
liposome
Fig. 6 Manipulation of cells and liposome using optical tweezers.
Issues/Barriers
Manipulation of cells and liposome using optical tweezers
Can we actually manipulate the nanoparticles and place them where we want them?
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• Measurement ChallengeMeasurement Challenge• Technical StrategyTechnical Strategy• Issues and BarriersIssues and Barriers• Dissemination of ResultsDissemination of Results• MilestonesMilestones• Resources and LeveragesResources and Leverages
Outline
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• Direct interactions with manufacturers such as FEI Co., Gatan and others
• Alliances with FDA and NIH
– Nanotechnology Characterization Laboratory
• Addresses several of the NNI Program Component Areas
– Reported through the NNI
• Publications and presentations
• Workshops and conferences
• Web-based tools for modeling and computation
Technology Transfer and Dissemination
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Measures of Success: Milestones
• Intercomparison of metrology techniques• Test particle uptake in cell cultures
2006__
2007__
2008__
2009__
2010__
Demonstration of dual beam sectioning Initial nanoparticle transport model
• Demonstration of cryomicroscopy capabilities• Demonstration of a robust measure of dispersion
• Demonstration of 3D reconstruction for biomedical applications• 3D chemical and dimensional metrology of test nanoparticles
• Development of a 3D particle nanometrology infrastructure
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Summary
This Competence at NIST will provide:• Development of an innovative toolbox for metrology of nanoparticles in biological systems
– Currently does not exist
• A strong group of experts in biological sample preparation
– Unbiased source of expertise
• Unique technical support for other agencies
– Make NIST THE place where other agencies come FIRST
GOAL:Develop a 3D particle nanometrology
infrastructure by 2010
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• Measurement ChallengeMeasurement Challenge• Technical StrategyTechnical Strategy• Issues and BarriersIssues and Barriers• Dissemination of ResultsDissemination of Results• MilestonesMilestones• Resources and LeveragesResources and Leverages
Outline
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Resource Requirements
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Leverages
• Nanotechnology Characterization Laboratory collaboration in particle characterization and standardization
– Postek, Vladar, MEL
– Scott, Small, CSTL
– Hackley, MSEL
• Food and Drug Administration collaboration in nanoparticle coatings– Karim, MSEL
• ATP Intramural Project
– Small, Postek
– Postek, Small
• 3-D Chemical Imaging Competence
– Scott, Small
• University of Maryland Laboratory for Ultrastructure Research
• FEI Company
• Hitachi High Technologies
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Thank you
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Thank you