1.  Nano Discovery Inc. Copyright Nano Discovery Inc. www.nanodiscoveryinc.com NANO

2. Nanoparticle-Enabled Dynamic Light Scattering Assay Gold Nanoparticle (AuNP) AuNP-antibody immunoprobe Immunoprobe bound with target protein Immunoprobe bound with target protein complex ~100 nm ~120 nm ~130-150 nm >>130-150 nm Target protein is detected by monitoring the nanoparticle size change!

3. Step I. Add sample to the nanoparticle probe solution Step II. Incubate the assay solution Step III. Measure the particle size change of the assay solution Average particle size (nm) Before assay After assay Intensity distribution Average particle size Protein concentration Measure by DLS Extract analyte information Add sample 100 nm150 nm All you need to do is to add the sample to the AuNP probe solution! !!

4. Equipped with an automatic 12-sample holder carousel Automatic measurement of 12 samples in multiple sequences High throughput capability (120-180 samples/hour) Maximum flexibility: assay formats and number of samples can be easily adjusted to fit into small and large scale studies Kinetic protein-protein binding study of 12 samples simultaneously Requires 40 µL of AuNP probe solution and 1-5 µL of sample solution Extremely easy-to-learn and easy-to-use software Instrument hardware is maintenance-free

5.  All following applications were based on reported, peer-reviewed scientific publications

6. Reference: 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. Average particle size increase (nm) Protein concentration or incubation time target protein binding partner of the target protein + Association and dissociation rate constant, k a and k d, can be determined. Method described in the NDS-1200 User Manual

7. NanoDLSay™  Homogeneous solution assay  Significantly faster reaction kinetics  Small sample volume (1-2 µL)  Monitor up to 12 kinetic binding studies simultaneously using the NDS-1200 system with maximum user flexibility SPR  Heterogeneous solid-liquid interface assay  Slow reaction kinetics  Large sample volume (10s-100s µL)  Limited to 1-2 kinetic binding studies. SPR instrument cost increases sharply for automatic study of larger number of samples Add sample Homogeneous solution assay Sample solution flow through SPR substrate: gold monolayer film

8. Reference: 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; 28: 8, 10-12. Average particle size increase (nm) Protein concentration or incubation time Matched antibody isotype Anti-isotype AuNP probe Unmatched antibody isotype Minimum size increaseSubstantial size increase

9. Average particle size increase (nm) Incubation time Maximum size increase  2 D (diameter of protein ) A size increase substantially larger than 2D indicates the presence of protein complex Reference: Jaganathan S, Yue P, Paladino DC, Bogdanovic J, Huo Q, Turkson J. A functional nuclear epidermal growth factor receptor, Src and Stat3 heteromeric complex in pancreatic cancer cells. PLoS One 2011, 6(5):e19605 (open access).

10. c Average particle size increase (nm) Incubation time Antibody screening c Particle size increase following antibody addition to the assay solution Conclusion: Protein and are binding partners of

11. NanoDLSay™  A simple two-step process that is completed in 10s of minutes or less  Requires only 1-2 µL of sample solution  Avoid non-specific interactions caused by centrifugation/isolation process  Reveal the “size” of the protein complex directly from the assay Co-immunoprecipitation  Multiple-step process that takes hours to days to complete  Requires 100s 1-2 µL of sample solution  Introduce substantial non-specific interactions during centrifugation/isolation process  No information regarding the “size” of the protein complex is revealed Step2. Analyze the binding partner Step1. Catch the target No isolation between step 1 and 2 Step1. Catch Step 2. Centrifuge/Isolate Step 3. Detach protein from the beads Multiple-steps: Immunoblotting

12. Particle size distribution curve (nm) Protein monomer and small complex: Smaller nanoparticle size increase Monodispersed particle size distribution Intensity distribution Protein aggregates: Substantially larger nanoparticle size increase Much broader particle size distribution curve 1. Bogdanovic J, Colon J, Baker C, Huo Q. A label-free nanoparticle aggregation assay for protein complex/aggregate detection and analysis. Anal. Biochem. 2010; 45:96-102. 2. Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:259-265.

13.  Traditional techniques such as analytical ultracentrifugation (AU), size exclusion chromatograph (SEC) are only suitable for studying purified protein solutions. For these techniques to work on real biological samples, labeling of the target protein is required  Fluorescence techniques require the labeling of the target proteins  Dynamic light scattering, although have been used for protein aggregate detection and study, is only suitable for high concentration protein solutions. DLS alone also cannot be used for protein aggregate detection from un-purified biological samples and fluids  Potential link between protein aggregation and cancer was first revealed using NanoDLSay™  Most recent scientific study has brought further evidence on the important role of protein aggregation in cancer NanoDLSay™ enables researchers to discover new molecular information associated with human diseases that have not been revealed using other existing techniques

14. NANO Nano Discovery Inc. 12565 Research Parkway Suite 400 Orlando, FL 32826 www.nanodiscoveryinc.com Tel: 407-770-8954 Email: huo@nanodiscoveryinc.comhuo@nanodiscoveryinc.com Copyright Nano Discovery Inc. June 2011 Disclaimer: 1. Patent application pending on NanoDLSay™ technology: PCT/US09/030087 and PCT/US11/21002 2. Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay™ technology