1. NanoDLSay TM – A Powerful Tool for Nanoparticle, Nanomedicine, Biomolecular and Pharmaceutical Research www.nanodiscoveryinc.com February 2010 Copyright of Nano Discovery, Inc.

2. What is NanoDLSay TM : 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 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y and Protein analyte Gold nanoparticle-antibody conjugates Assay Procedure Add sample Incubate Measure by DLS Before Assay Distribution 50 100 Average particle Size (nm) After Assay Nanoparticle clusters February 2010 Copyright of Nano Discovery, Inc.

3. What is Dynamic Light Scattering (DLS)? Average particle size (nm) 50 100 150 Intensity Distribution 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 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 February 2010 Copyright of Nano Discovery, Inc.

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 10 5 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 Dark field optical images of GNPs mixed with human serum (A) and PS particles (B) and nanorod (C)c GNPs Serum A GNPs PS particle B C Gold nanorods February 2010 Copyright of Nano Discovery, Inc.

5.  Obtain results in minutes instead of hours and days  Easy to conduct (a one-step process!)  Almost labor-free, no special training needed  Simple instrument ($40-60K, instead of $100sK +)  Low cost and high sensitivity  Can be easily adapted for protein panel analysis  Extensive range of applications Unique Features of NanoDLSay  ELISA: takes hours to days to conduct the assay  Western blot: takes days to complete and labor-intensive  Surface plasmon resonance: too expensive ($200-500K)  Applications: limited Advantages Over Traditional Techniques February 2010 Copyright of Nano Discovery, Inc.

6. 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 2010 Copyright of Nano Discovery, Inc.

7. Broad Applications of NanoDLSay TM  General assay for protein detection & analysis  Protein-protein interaction study  Biomolecular binding kinetics study  Antibody isotyping and quality control analysis  Protein complex study and analysis  Protein aggregation detection and 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 bioconjugate quality control analysis February 2010 Copyright of Nano Discovery, Inc.

8. Application 1. As a General Sandwich Immunoassay for Protein Detection Huo, et al. JACS, 2008, 130, 2780-2782 Huo, et al. J. Immunol. Method 2009, 349, 38-44. Average particle size (nm) Target protein concentration + + + + + Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y and Target protein 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 February 2010 Copyright of Nano Discovery, Inc.

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, 9425-9432 Average particle size (nm) Incubation Time (min) Average particle size (nm) Antibody Concentration (µg/mL) In-situ monitoring of the adsorption process Antibody concentration effect study February 2010 Copyright of Nano Discovery, Inc.

10. 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 Y Y No interaction No size increase Huo, et al. Anal. Chem. 2009, 81, 9425-9432 Huo, et al. American Biotechnology Laboratory 2010, in press Average particle size (nm) 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.

11. 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: Size distribution curve Relative Intensity Size distribution (nm) Polydispersed Monodispersed Average particle size (nm) Target protein concentration + + Dose-Response Curve

12. 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 Beta 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

13. For Further Information Contact: Nano Discovery Inc. Tel: 407-770-8954 Email: huo@nanodiscoveryinc.com www.nanodiscoveryinc.com 12565 Research Parkway Suite 300, Orlando, FL 32826