NanoDLSayTM – A Powerful Tool for Nanoparticle, Nanomedicine,

Biomolecular and Pharmaceutical Research

February 2010 Copyright of Nano Discovery, Inc.

What is NanoDLSayTM: Gold Nanoparticle (GNP) Coupled with Dynamic Light Scattering (DLS) for

Biomolecular Assay

Nanoparticle bioconjugate

• Gold nanoparticle bioconjugates interact with the analyte to form a nanoparticle cluster. • The nanoparticle size increase, detected by DLS, is correlated to analyte concentration

Nanoparticle clusters: size increase

Y

Y

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

Y

Yand

Protein analyte

Gold nanoparticle-antibody conjugates

February 2010 Copyright of Nano Discovery, Inc.

Assay Procedure

Add sample

Incubate Measureby DLS

Before Assay

Distribu

tion

50 100Average particle Size (nm)

After AssayNanoparticle clusters

What is Dynamic Light Scattering (DLS)?

Average particle size (nm)50 100 150

Inte

nsity

Dis

trib

utio

n Particle size and size distribution• Typically two types of data are obtained from DLS

measurement: one is the average particle size and one is 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

February 2010 Copyright of Nano Discovery, Inc.

DLS: An instrument for particle size measurement

Sample solution

Laser beam

Detector

Correlator

• 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

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

Figure: Dark field optical images of GNPs mixed with human serum (A) and PS particles (B)

GNPs

Serum

A

GNPs

PS particle

B C

Gold nanorods

February 2010 Copyright of Nano Discovery, Inc.

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

Unique Features of NanoDLSayTM

February 2010 Copyright of Nano Discovery, Inc.

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 ($200-500K) Applications: limited

Comparison with Traditional Techniques:

Various Types of Gold Nanoparticle Size Change Upon Binding with Target Protein

Molecules

February 2010 Copyright of Nano Discovery, Inc.

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!

Broad Applications of NanoDLSayTM

February 2010 Copyright of Nano Discovery, Inc.

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

Application 1. As a General Sandwich Immunoassay for

Protein Detection

Huo, et al. JACS, 2008, 130, 2780-2782Huo, et al. J. Immunol. Method 2009, 349, 38-44.

Ave

rage

par

ticle

siz

e (n

m)

Target protein concentration

++

+

+

+

Y

Y

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

Y

Yand

Target protein

February 2010 Copyright of Nano Discovery, Inc.

• 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

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, 9425-9432

Ave

rage

par

ticle

siz

e (n

m)

Incubation Time (min)

Ave

rage

par

ticle

siz

e (n

m)

Antibody Concentration (µg/mL)

In-situ monitoring of theadsorption process

Antibody concentration effect study

February 2010 Copyright of Nano Discovery, Inc.

Application 3. Protein-Protein Binding and

Interaction Study• 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

Y

Y

Y

Y

Y

Y

YY

No interactionNo size increase

Huo, et al. Anal. Chem. 2009, 81, 9425-9432Huo, et al. American Biotechnology Laboratory 2010, in press

Ave

rage

par

ticle

siz

e (n

m)

Incubation Time (min)

+

+

++

++ +

Y

Y

Y

Y

Y

Y

Y

Y

Matching target protein

Y

Y

Y

Y

Y

Y

Y

Y

February 2010 Copyright of Nano Discovery, Inc.

Application 4. Detect Protein Complex/Aggregate

Formation

February 2010 Copyright of Nano Discovery, Inc.

• 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

How to identify protein complex/aggregate formation from NanoDLSay analysis:

Ave

rage

par

ticle

siz

e (n

m)

Target protein concentration

+

+

Size distribution curve

Rel

ativ

e In

tens

ity

Size distribution (nm)

Polydispersed

MonodispersedDose-Response Curve

1) 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, 9425-9432

2) 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

3) 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, 38-44.

4) 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, 8138-8139.

5) 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, 2780-2782. (also featured at JACS Select #5, 2009, free)

6) 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, 9668-9671.

Examples: Refer to Publications

For Further Information

Contact:

Nano Discovery Inc.Tel: 407-770-8954Email: huo@nanodiscoveryinc.comwww.nanodiscoveryinc.com

12565 Research Parkway Suite 300, Orlando, FL 32826