One new technology, discover a new world NanoDLSay™ for Protein-Protein Interaction Study, Label-free Protein Complex and Protein Oligomer/Aggregate Detection and Analysis in Real Biological Samples Copyright Nano Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954 Email: huo@nanodiscoveryinc.com

Traditional immunoassay assumes proteins only exist as monomer! A fundamental problem with the current bioassay In real biological systems, a biomolecule exists not only as individual molecules, but also as complexes, and this issue is severely neglected by the current assay techniques Individual proteins Protein complexes Protein aggregates Traditional immunoassay assumes proteins only exist as monomer! I. We will not know if any complex is here or not The complex leads to a fake lower concentration II. The labeled signaling antibody cannot recognize the complexed target protein Conclusion in this case: target protein is not present in the sample at all!!! III.

What is NanoDLSay™: Detect target proteins by monitoring the size change of nanoparticles upon binding with the target protein Immunoglobulin G (IgG) (~ 7-10 nm) Protein monomer (~ 5-20 nm) Protein complex (>> 5-20 nm) D = 100 nm Gold nanoparticles (AuNP) D ~ 120 nm D ~ 130-160 nm D >> 130-160 nm

Dynamic light scattering (DLS): Measure particle size in nanometer size range Scattered light intensity fluctuation Laser beam Scattering light Correlation function Large particle Small particle Intensity Distr. (%) 100 120 200 Average particle size (nm)

Protein-AuNP interactions: Au-S, Au-N bonding, electrostatic and van der Waals interaction “Protein Corona” References 1. Dobrovolskaia MA, Patri AK, Zheng J, Clogston JD, Ayub N, Aggarwal P, Neun BW. Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles. Nanomedicine (Nanotechnology Biology and Medicine) 2009, 5, 106-117. 2. Lacerda SHDP, Park JJ, Meuse C, Pristinski D, Becker ML, Karim A, Douglas JF. Interaction of gold nanoparticles with common human blood proteins. ACS Nano 2010, 4, 365-379. 3. Calzolai L, Franchini F, Gilliland D, Rossi F, Protein—nanoparticle interaction: identification of the ubiquitin-gold nanoparticle interaction site. Nano Lett. 2010; 10: 3101-3105.

Why not to measure the size of the biomolecules directly using DLS? The scattering light intensity of biomolecules is too weak Such analysis can be done, but at very high concentration – not reflecting the true state of a protein in biological samples Such analysis can only be done on pure protein samples

Why Gold Nanoparticles (AuNPs)? Exceptionally intense light scattering property 105 times stronger than a fluorescent dye molecule; 100s-1000s times stronger than polystyrene (PS) latex particles Detection limit of DLS for AuNPs can easily reach fM to aM range As an optical probe, AuNPs easily stands out from sample matrix AuNPs Serum A PS particle B C Gold nanorods Dark field optical images of AuNPs mixed with human serum (A) and PS particles (B) and gold nanorod AuNR (C)

NanoDLSay™: Detect target proteins in all forms At a saturated binding, the average particle size increase of the assay, , is approximately twice of the diameter (D) of the protein A protein complex is typically larger than a monomer A protein complex causes larger average particle size increase of the assay than a protein monomer When a protein exists as an oligomer aggregate, it may crosslink the nanoparticles into clusters, leading to a substantial particle size increase of the assay Applicable for detecting other biomolecule complexes such as DNA-protein complexes 3 Average particle size increase (nm) 2 1  = 2D of analyte Information on the target molecule is obtained from real biological samples 0 min Incubation time (min) 30 min

Applications Real-time kinetic binding study of protein- protein interaction Label-free protein complex detection and analysis in real biological samples Label-free protein oligomer and aggregate detection and analysis in real samples Comparison of NanoDLSay™ with other existing techniques

I. Kinetic study of protein-protein interaction Assay format I: immobilize one target protein on AuNP as a probe Procedure: Immobilize one target A or B protein to the AuNP Mix the target A or B-modified AuNP with target B or A protein Monitor the AuNP size change Binding affinity may be estimated using Langmuir adsorption model Target A Target B Average particle size (nm) Requirements: Immobilization of A or B on AuNP does not affect protein-protein interactions Non-binding proteins Sample suitability: Pure protein samples Incubation time (min) 30 Homogeneous solution assay, obtain results in minutes Detect both strong and weak binding Monitoring binding in real-time

I. Kinetic study of protein-protein interaction Assay format II: allow proteins bind first in solution, then conduct adsorption assay + + A B C complex Comparison to obtain binding information Procedure: Mix relevant protein binding partners together Conduct adsorption assay of individual target with AuNP Conduct adsorption assay of mixed product with AuNP Comparison of assay results from 2 and 3 to obtain complex information Requirements: At least one protein will readily adsorb to AuNP Sample suitability: Pure protein samples Compared to assay format I: The AuNP-adsorption assay does not affect target protein binding Suitable for studying multi-binding partner (more than 2) complexes

I. Kinetic study of protein-protein interaction Assay format III: two AuNP probe interaction assay Target A Target B + Limitations: Immobilization of A or B on AuNP does not affect protein-protein interactions Procedure: Make a target A or B-conjugated AuNP probe Mix the two probes in solution Monitor the size change of the assay A-B interaction leads to AuNP cluster formation Sample suitability: Pure protein samples Compared to assay format I and II: Increase assay sensitivity for detecting weak interactions Not limited to the size of the proteins or other target molecules

Jans H, Liu X, Austin L, Maes G, Huo Q 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; 28: 8, 10-12. Among six monoclonal antibodies from a vendor X, three have quality problem Surface plasmon resonance works on this application, but expensive Assay kit from vendor Y costs $20 per analysis, NanoDLSay costs 20-30¢ per assay

Particle size change upon antibody addition II. Label-free protein complex detection and binding partner analysis from real samples Step 2: Binding partner screening using antibody Step 1: Catch the target Average particle size increase (nm) c Binding partners Particle size change upon antibody addition Not binding partners  ~ 2D Incubation time (min) Step 1. Determine if a target protein exists as a complex (The final net increase of the AuNP size tells how big the target protein is) Step 2. Screen and identify the binding partners to the target protein

Jaganathan, S. ; Yue, P. ; Paladino, D. C. ; Bogdanovic, J. ; Huo, Q Jaganathan, S.; Yue, P.; Paladino, D.C.; 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). Step 1: Catch the target Experiments: Prepare the AuNP immunoprobe for EGFR Use the EGFR-AuNP probe to catch the target Determine if EGFR is in a complex Control: add anti-EGFR to the sample solution, incubate, and then repeat the binding assay with AuNP immunoprobe. AuNP immunoprobe for target protein, EGFR EGFR is about 10-15 nm, 70 nm of increase suggests it is a complex! ~ 70 nm Control study

Screening the binding partners in the complex using specific antibody Step 2: Binding partner screening using antibody Experiments: Add an antibody for the suspected binding partner into the assay solution If the particle size is increased, then it is a positive response, and vice versa Control: add a negative isotype control antibody to the assay solution If we conduct a sandwich assay, the conclusion will be: EGFR is not there! Negative control

III. Label-free protein oligomer/aggregate detection and analysis Average particle size increase (nm) Incubation time (min)  = 2D of analyte 0 min 30 min protein monomer oligomers, aggregates Protein oligomer/aggregates cause AuNP probe cluster formation Specific detection of target protein oligomer/aggregates in real samples

Bogdanovic J, Colon J, Baker C, Huo Q 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.

Detection of human IgG dimer and discovery of a new molecular test for prostate cancer diagnosis using IgG-AuNP adsorption assay Huo, Q.; Litherland, S.A.; Sullivan, S.; Hallquist, H.; Decker, D.A.; Rivera-Ramirez, I. Developing a nanoparticle test for prostate cancer scoring. J. Translational Medicine, 2012, 10:44 (open access). Citrate-AuNP D ~ 100 nm IgG dimer IgG Average D ~ 300 nm NanoDLSay reveals human IgG dimerization at > 100 µg/mL Tumor-IgG interaction reflected in the IgG-AuNP adsorption assay

Comparison of NanoDLSay™ with other existing techniques

NanoDLSay™ versus Surface Plasmon Resonance (SPR) Label-free technique Optical substrate: gold nanoparticle Read-out: AuNP size change Homogeneous solution assay Low cost of consumables Reveal the size information of the target analyte, distinguish protein complexes and oligomers/complexes from monomers Label-free technique Optical substrate: gold thin film Read-out: refractive index change Heterogeneous chip assay High cost of consumables Does not reveal the size information of the target analyte, does not tell whether a protein is a monomer, complex or oligomer

Comparison of NanoDLSay™ with co-immunoprecipitation (Co-IP) followed by immunoblotting for protein complex analysis

NanoDLSay™ versus size exclusion chromatography (SEC) and analytical ultra-centrifugation (AU) for protein complex and oligomer/aggregate detection and analysis SEC and AU: For pure protein solution study only SEC underestimates complex or oligomer/aggregate formation (eluent dilution disrupts existing complexes/oligomers) AU overestimates complex or oligomer/aggregate formation (centrifugation artificially increases protein complexes/oligomers) NanoDLSay™: Detect protein complexes, oligomers/aggregates from real samples Fast screening test for protein complex/oligomer/aggregates

Non-specific interactions: effect on Co-IP and NanoDLSay™ A problem in Co-IP: Significant non-specific interactions caused by the separation process The concentration of the particle probes and proteins is artificially increased during centrifugation, increasing non-specific interactions This problem does not exist in NanoDLSay™: The AuNP probe concentration is relatively low, reducing non-specific interactions No centrifugation separation is involved

Product & Services NDS1200: A new dynamic light scattering instrument designed for performing NanoDLSay™ Automatic measurement of 12 samples Automatic kinetic study of 12 samples Fast analysis time: 10-20s per sample 40 µL assay solution is used for the measurement Low-cost, disposable min-glass tubes with caps are used as sample containers. No cross-contamination between samples High throughput analysis capability: 120-180 samples/hour The hardware is maintenance-free No special housing environment is required for the instrument Extremely easy-to-use software

Product & Services NanoDLSay™ software: A software designed for flexible, kinetic and high throughput analysis

Product and Order Information NDS1200 Dynamic light scattering instrument for conducting NanoDLSay™ NDS-Kit1000 Assay kit including disposable sample cells and other consumables Please Contact Us to Request a Quote: 3251 Progress Drive Suite A1 Orlando, FL 32826 Phone: 407-770-8954 Email: sales@nanodiscoveryinc.com Or visit online: www. nanodiscoveryinc.com Notes Patent application pending on NanoDLSay™ technology and NDS1200 system: PCT/US09/030087 and PCT/US11/21002 Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay™ technology