1. NanoDLSayTM – A Powerful Tool for Nanoparticle, Nanomedicine, Biomolecular and Pharmaceutical Research
Nano Discovery, Inc
February Copyright of Nano Discovery, Inc.

2. Average particle size increase
What is NanoDLSayTM: Gold Nanoparticle Coupled with Dynamic Light Scattering (DLS) for Biomolecular Assay Y Y Y
and
antigen
Gold nanoparticle-antibody conjugates
Average particle size increase
Assay Procedure
Before Assay
Distribution 50
100
Average particle Size (nm)
After Assay
Add
sample
Incubate
Measure
by DLS
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3. What is Dynamic Light Scattering (DLS)?
Sample solution
Laser beam
Detector
Correlator
DLS: An instrument for particle size measurement
DLS detects the scattered light from the particles in the sample solution
Particles in the solution undergo a constant Brownian motion
The Brownian motion of a larger particle is slower than a smaller particle. This is how DLS measures the particle size of a sample
The scattered light intensity fluctuation detected by DLS is converted into particle size information through a correlator
Typically two types of data are obtained from DLS measurement: one is the average particle size and one the particle size distribution curve.
The average particle size tells you what is the average size of all the particles in the sample
The size distribution curve tells you what is the relative distribution of each group of particles, if polydispersed particles are present
Both types of data are used in NanoDLSay to extract information on the target biomolecules
Average particle size (nm) 50
100
150
Intensity Distribution
Particle size and size distribution
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4. Why Gold Nanoparticles (GNPs)?
Gold nanoparticles (GNPs) have exceptionally large light scattering cross section at or near their surface plasmon wavelength region
Gold nanoparticles scatter light 105 times stronger than a fluorescent dye molecule; 100s times stronger than polystyrene (PS) latex particles
The scattered light of gold nanoparticles does not suffer from the photobleaching often encountered in fluorescent molecules
Detection limit of DLS for GNPs can easily reach fM to aM range
GNPs
Serum A
PS particle B C
Gold nanorods
Figure: Dark field optical images of GNPs mixed with human serum (A) and PS particles (B)
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5. Unique Features of NanoDLSayTM
Obtain results in minutes instead of hours and days
Easy to conduct (a one-step process!)
Minimum labor involved in the assay
Simple instrument (DLS instrument costs $40-60K )
Low cost and high sensitivity
Can be easily adapted for protein panel analysis
Extensive range of applications
Comparison with Traditional Techniques:
ELISA: takes days to prepare and hours to conduct the assay
Western blot: takes days to complete, labor-intensive, special training
Surface plasmon resonance: too expensive ($ K)
Applications: limited
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6. Particle size increase
Various Types of Gold Nanoparticle Size Change Upon Binding with Target Protein Molecules
Sandwich assay of protein or protein complex
Protein-protein interaction study
Protein complex/ aggregate detection
Particle size increase
Different particle size increase is used for different assay applications!
February Copyright of Nano Discovery, Inc.

7. Broad Applications of NanoDLSayTM
General assay for protein detection & analysis
Protein-protein interaction study
Biomolecular binding kinetics study
Receptor-ligand identification
Antibody isotyping and quality control analysis
Protein complex analysis
Protein aggregation study
Detection of non-protein biomolecules
Detection of small chemicals and ions
Protein inhibitor screening and drug development
Biopharmaceutical research and development
Detection of viruses and bacteria
Nanoparticle bioconjugate development
Nanoparticle quality control
Nanoparticle size analysis
February 2010
Copyright of Nano Discovery, Inc.

8. Application 1. As a General Sandwich Immunoassay for Protein Detection
Target protein
Average particle size (nm)
Target protein concentration +
Two different monoclonal antibodies are conjugated to two different GNPs
Sandwich structure formation between the target protein and two GNP probes will cause nanoparticle cluster formation, therefore, lead to the average particle size increase of the assay solution
The average particle size increase can be correlated to target protein concentration
Two monoclonal antibodies may be replaced by a polyclonal antibody
Huo, et al. JACS, 2008, 130,
Huo, et al. J. Immunol. Method 2009, 349,
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9. Application 2. Monitor Gold Nanoparticle Bioconjugation Process and Quality Control
Nanoparticle size increase
An extremely powerful tool to monitor gold nanoparticle bioconjugation process
Analyze the quality, stability and binding affinity of gold nanoparticle bioconjugates
Huo, et al. Anal. Chem. 2009, 81,
Average particle size (nm)
Incubation Time (min)
Antibody Concentration (µg/mL)
In-situ monitoring of the
adsorption process
Antibody concentration effect study
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10. Application 3. Protein-Protein Binding and Interaction Study
Matching target protein
In-situ monitoring of protein-protein binding and interaction study
When target protein binds to protein conjugated to gold nanoparticles, the particle size will increase
A function very similar to the Surface Plasmon Resonance technique
Example 1: can be used to confirm the binding affinity of bioconjugated gold nanoparticles
Example 2: can be used for antibody isotyping and quality control analysis
Average particle size (nm)
Incubation Time (min) + Y
No interaction
No size increase
Huo, et al. Anal. Chem. 2009, 81,
Huo, et al. American Biotechnology Laboratory 2010, in press
February Copyright of Nano Discovery, Inc.

11. Application 4. Detect Protein Complex/Aggregate Formation
How to identify protein complex/aggregate formation from NanoDLSay analysis:
Nonlinear increase of nanoparticle size at a critical target protein concentration
The particle size increases dramatically and quickly at this critical concentration
Particle size distribution curve often reveals very broad and multi-model polydispersed distribution. Run-to-run variation is often large
Size distribution curve
Dose-Response Curve
Relative Intensity
Size distribution (nm)
Monodispersed +
Polydispersed
Average particle size (nm) +
Target protein concentration
February Copyright of Nano Discovery, Inc.

12. Examples: Refer to Publications
Jans, H.; Liu, X.; Austin, L.; Maes, G.; Huo, Q. Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding study. Anal. Chem. 2009, 81,
Austin, L.; Liu, X.; Huo, Q. An immunoassay for monoclonal antibody isotyping and quality analysis using gold nanoparticles and dynamic light scattering. American Biotechnology Laboratory 2010, in press
Liu, X.; Huo, Q. A washing-free and amplification-free one-step homogeneous assay for protein detection using gold nanoparticle probes and dynamic light scattering. J. Immunol. Method 2009, 349,
Dai, Q.; Liu, X.; Coutts, J.; Austin, L.; Huo, Q. A one-step highly sensitive method for DNA detection using dynamic light scattering. J. Am. Chem. Soc. 2008, 130,
Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo, Q. A One-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering. J. Am. Chem. Soc. 2008, 130, (also featured at JACS Select #5, 2009, free)
Ray, P.C. et al. Use of gold nanoparticles in a simple colorimetric and ultrasensitive dynamic light scattering assay: selective detection of arsenic in groundwater. Angew. Chem. Int. Ed. 2009, 48,

13. For Further Information
Contact:
Nano Discovery Inc.
Tel:
Nano Discovery, Inc
12565 Research Parkway Suite 300, Orlando, FL 32826