1. Metrology for the “Fate” of Nanoparticles in Biosystems
Michael T. Postek,
Andras Vladar, Thomas LeBrun and John Dagata, MEL
John Small,
John Henry Scott, Scott Wight, Peter Barker, CSTL
Alamgir Karim,
Jack Douglas, Matthew Becker, Vincent Hackley,
Andrew Allen, Stephanie Hooker, MSEL

2. MEL MSEL CSTL Metrology for the “Fate” of Nanoparticles
Michael T. Postek, Champion
Andras Vladar
Thomas LeBrun
John Dagata,
Alamgir Karim, Champion
Jack Douglas
Matthew Becker
Vincent Hackley
Andrew Allen
Stephanie Hooker
John Small, Champion
John Henry Scott
Scott Wight,
Peter Barker

3. Metrology for the “Fate” of Nanoparticles
Competence proposal is motivated by work stemming from:
National Nanotechnology Initiative
Subcommittee on Nanoscale Science, Engineering and Technology (NSET)
Michael Postek
Alamgir Karim
Nanotechnology Environmental and Health Implications Working Group (NEHI WG)
John Small
The National Institute for Occupational Safety and Health (NIOSH)
Andrew Maynard (endorsement)
Environmental Protection Agency (EPA)
Barbara Karn
Nora 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

4. Why Now…Impact on Society, Environment,
and Commerce
NIST is the only organization that has the mission and the necessary combination of
staff expertise, experience, industry contacts, industry respect, & worldwide recognition
as 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.
Without NIST’s contributions…..
the lack of a common infrastructure for bioscience measurements, standards, & data will
constrain innovation and stifle the commercialization of new bioscience-based applications
in 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 for 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.

5. X Flawed Science Big PRESS
Why Now...Potential for Catastrophic Effects:
Psychological and Economic
NANOTECHNOLOGY 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 X
Flawed Science Big PRESS
This approach will not reassure the public at large
For Science, Nanotech Poses Big Unknowns
By Rick Weiss Washington Post Staff Writer Sunday, February 1, 2004; Page A01
Materials Today
July/Aug 2003: 64
Animal studies are mixed
Polish study found little effect
U.S. study found many rats died early and lung granulomas
Wing Lam of Wyle Laboratories in Houston
David Warheit at DuPont Co.'s Haskell Laboratory (15% died within 24 hours!)
Appears more toxic than quartz.
What materials were used?
Asbestos diseases often take over 20 years to show up!

6. NIST’s Role Why NIST…Lack of Measurement Infrastructure has
Become a Critical Hurdle to Further Innovation
“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.
VALUE CREATION (IMPACT): Productivity / Market Access / Public Safety & Welfare
March 2004
Standards
Development
(Phase II)
Measurement Methods & Tools
(Phase I)
Basic Science
NIST’s
Role
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.
Scientific
Discovery
New
Invention
Period
Technology
Improvement
Period
S&T DEVELOPMENT OVER TIME
*Office of Cellular, Tissue, and Gene Therapies

7. It is this simple - what is the difference?
Micrometer sized particles
of Zinc Oxide
Left
Right
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
What is the effect on the body?
Right
Nanometer sized particles
of Zinc Oxide
Primary Particle size of 3-10 nm

8. Competence Goal What we want to do: What we are not doing:
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

9. Why Nanoparticle Metrology for Biological Systems?
Motivation
Why Nanoparticle Metrology for Biological Systems?
Nanoparticles are being produced and are a part of our environment
Nanoparticle measurements are challenging
Biological samples must be carefully prepared
Validation procedures are needed for standardization of measurements
NIST needs to develop competence in this area
NIST can make a difference
Why 3D chemical imaging?
New technologies are creating more characterization problems that cannot be solved by traditional 2D and point analysis methods
These problems are found over a wide variety of current and future nanotechnologies including:
semiconductors, optoelectronics, data storage, catalysts, pigments, nano-composites, powder metallurgy and ceramics, wear-resistant and thermal coatings, biomaterials, etc.
Many devices and components are now smaller than our analytical sampling volume.
Requires deconvolution of matrix or adjacent components
3D analysis is the best approach to solve this problem
Why now?
New enabling technologies:
Electron microscope optics, detectors, stage automation, computing power, instrument stability

10. Outline
Measurement Challenge
Technical Strategy
Issues and Barriers
Dissemination of Results
Milestones
Resources and Leverages

11. Outline
Measurement Challenge
Technical Strategy
Issues and Barriers
Dissemination of Results
Milestones
Resources and Leverages

12. Nano-particle Life-Cycle
Underlying Basis for Measurement Need
Effect of size
Exposure/Uptake
Bioactivity
Transport
Nano-particle
Life-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
Control of mobility and distribution
Surface chemistry effects

13. Challenge: Biological Sample Preparation
Biological samples require:
Fixation
Dehydration
Embedding
Sectioning
Dual beam FIB
Cryomicroscopy
Cryomicrotomy
Provides a new paradigm for NIST
Physical metrology
Biological sample preparation is not new to NIST, but not extensively practiced at NIST
Biological samples require expertise that must be developed at NIST in order to assist our customers in metrology related situations.

14. Nanotubes (C, Au, Hybrid…)
Challenge: Diversity of Nanoparticles Presents Numerous Characterization Challenges
Dendrimers
Quantum Dots
Nanotubes (C, Au, Hybrid…)
SWNT
DWNT
MWNT
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
Micelles
Liposomes
Functionalized
Nanoparticles

15. Challenge: Numerous Measurement Parameters of Nanoparticles
particle diameter
core diameter
Core composition
&
stiffness
shell thickness
Molecular mass
& distribution
Surface chemistry
&
Extent of bio-functionality
Aggregation number
(# molecules per particle)
Dispersion
Properties Impacted by Measurable Parameters
pH susceptibility
Bioactivity
Diffusion coefficient
Anisotropic transport
Membrane permeability
Degradation mechanisms and byproducts

16. Challenge: Increasing Complexity
Fullerene decorated
Dendrimer
AW Jensen, BS Maru, X Zhang, DK Mohanty, BD Fahlman, DR Swanson, and DA Tomalia
Nano Letters, 5(6), , 2005
Dendrimer decorated
Polymer Brush
Antibody labeled
Gold Nanoshell
CC Lee, M Yoshida, JMJ Frechet, EE Dy, and FC Szoka
Bioconjugate Chem., 16(3), , 2005
C Loo, A Lowery, N Halas, J West, and R Drezek
Nano Letters, 5 (4), , 2005

17. Small Changes Cause Enormous Differences in the Bioactivity
Challenge: High Measurement Precision of Surface Chemistry
Small Changes Cause
Enormous Differences in the Bioactivity
Variable surface chemistry induces
6 orders of magnitude
difference in toxicity profile
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), , 2004

18. Modeling is Imperative
Challenge: Multiple mechanisms of particle translocation
Modeling is Imperative
Computational approaches can help elucidate potential mechanisms of translocation

19. Outline
Measurement Challenge
Technical Strategy
Issues and Barriers
Dissemination of Results
Milestones
Resources and Leverages

20. Strategic Technical Approach
Bare
Nanoparticle
Characterization
Cell Culture
Sample Preparation
Flocculation Particle Tracking
Extra & Intra Cellular Nanoparticle Characterization
Transport Properties
Simulations Modeling

21. A Multi-laboratory Effort
Modeling
Microscopy
Cell Culture
Specimen
Preparation
Analytical

22. Initial Test Nanoparticles and Metrology
Extracellular Metrology
Intracellular Metrology
Cell Culture
Ultrastructure
Particle Characterization
TEM
TEM (sectioned material)
SEM
SEM (sectioned material)
FIB (sectioned material)
Cryomicroscopy
Dimensional Metrology
DLS
USAXS
Particle Imaging
Fluorescence
AFM
Transport Modeling x
Particle Placement
Optical Tweezers
Particle Localization
Particle Removal
Gold nanoshell
Quantum
Dot

23. A Multi-laboratory Effort
Modeling
Single Particle Analysis
and Manipulation
Microscopy
Transmission Electron Microscopy
NEW Aberration Corrected TEM
Potential New NIST competence
Scanning Electron Microscopy
NEW Environmental SEM
Optical Tweezers
Single nanoparticle manipulation
in biological materials
Atomic Force Microscopy of
biological materials
3-D reconstruction of
cells and particles
Nanometrology
Cell Culture
Specimen
Preparation
Analytical

24. A Multi-laboratory Effort
Modeling
Single Particle Analysis
and Manipulation
Microscopy
Transmission Electron Microscopy
NEW Aberration Corrected TEM
Potential New NIST competence
Scanning Electron Microscopy
NEW Environmental SEM
Optical Tweezers
Single nanoparticle manipulation
in biological materials
Atomic 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 Si K O
Nanometrology
Cell Culture
Specimen
Preparation
Analytical

25. A Multi-laboratory Effort
NOVA Nanolab - dynamic
sectioning of biological
material for
nanoparticle analysis
and metrology
Potential New NIST competence
Biological sample
preparation
for electron microscopy
Modeling
Microscopy
Nanometrology
Focused Ion Beam
NOVA Nanolab
Biological Sample Preparation
UMd
Cell Culture
Specimen
Preparation
Analytical

26. A Multi-laboratory Effort
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)
USAXS SLIT-SMEARED GEOMETRY
USAXS compliments the microscopy methods, providing
statistically relevant data on populations of particles under in situ conditions
Modeling
Microscopy
Nanometrology
Cell Culture
Specimen
Preparation
Analytical
X-ray Scattering

27. A Multi-laboratory Effort
Dh = Hydrodynamic diameter of SCK
Dynamic Light Scattering (DLS)
Modeling
Microscopy
Nanometrology
Cell Culture
Specimen
Preparation
Analytical
Dynamic Light Scattering

28. Outline
Measurement Challenge
Technical Strategy
Issues and Barriers
Dissemination of Results
Milestones
Resources and Leverages

29. Microscopy Measurements
Issues/Barriers
Microscopy Measurements
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)

30. Issues/Barriers Modeling
Numerous proposed mechanisms and controversy in the scientific literature motivates intensive experimental and computational study of these types of assembly processes.
Endocytosis and the Encapsulation of Nanoparticles by Clathrin
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
There is currently no systematic experimental or computational approach to predict how nanoparticles might affect normal biological processes.
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.

31. Manipulation of cells and liposome using optical tweezers
Issues/Barriers
Can we actually manipulate the nanoparticles
and place them where we want them?
Fig. 6 Manipulation of cells and liposome using optical tweezers.
Manipulation of cells and liposome using optical tweezers

32. Outline
Measurement Challenge
Technical Strategy
Issues and Barriers
Dissemination of Results
Milestones
Resources and Leverages

33. Technology Transfer and Dissemination
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

34. Measures of Success: Milestones
2006__
2007__
2008__
2009__
2010__
Intercomparison of metrology techniques
Test particle uptake in cell cultures
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

35. Develop a 3D particle nanometrology
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

36. Outline
Measurement Challenge
Technical Strategy
Issues and Barriers
Dissemination of Results
Milestones
Resources and Leverages

37. Resource Requirements

38. 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

39. Thank you

40. Thank you