1. NanoDLSay TM – A New Solution for Biomolecular Detection and Analysis February 2010 Copyright of Nano Discovery, Inc.

2. Biomolecular Detection and Analysis: the foundation of the whole modern life science research and medical diagnosis February 2010 Copyright of Nano Discovery, Inc.

3. What is NanoDLSay TM : Gold Nanoparticle Coupled with Dynamic Light Scattering (DLS) for Biomolecular Assay Add sample Incubate Measure by DLS Increasing antigen concentration Distribution 50 100 Size (nm) Assay Procedure 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 antigen Size increase Gold nanoparticle- antibody conjugates 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 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.

5. Dynamic Light Scattering (DLS) Scattered light intensity fluctuation Autocorrelate intensity fluctuations Sample solution Laser beam Detector Correlator Average particle size (nm) 50 100 150 Intensity Distribution Particle size and size distribution

6.  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 TM 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:

7. Various Types of Gold Nanoparticle Size Change Upon Binding with Target Protein Molecules February 2010 Copyright of Nano Discovery, Inc. Sandwich assay Protein-protein interaction study Protein complex/ aggregate detection Particle size increase

8. Broad Applications of NanoDLSay TM 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

9. Application 1. As a General Sandwich Immunoassay for Protein Detection Huo, et al. JACS, 2008, 130, 2780-2782 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 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

10. 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.

11. 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. 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.

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

13. 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

14. 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