NanoDLSayTM – A Powerful Tool for Nanoparticle, Nanomedicine, Biomolecular and Pharmaceutical Research Nano Discovery, Inc February 2010 Copyright of Nano Discovery, Inc.

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 February 2010 Copyright of Nano Discovery, Inc.

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 February 2010 Copyright of Nano Discovery, Inc.

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) February 2010 Copyright of Nano Discovery, Inc.

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 ($200-500K) Applications: limited February 2010 Copyright of Nano Discovery, Inc.

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 2010 Copyright of Nano Discovery, Inc.

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.

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, 2780-2782 Huo, et al. J. Immunol. Method 2009, 349, 38-44. February 2010 Copyright of Nano Discovery, Inc.

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) Antibody Concentration (µg/mL) In-situ monitoring of the adsorption process Antibody concentration effect study February 2010 Copyright of Nano Discovery, Inc.

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, 9425-9432 Huo, et al. American Biotechnology Laboratory 2010, in press February 2010 Copyright of Nano Discovery, Inc.

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 2010 Copyright of Nano Discovery, Inc.

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, 9425-9432 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, 38-44. 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. 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) 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.

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