One New Technology, Discover a New World NanoDLSay™: Nanoparticle-Enabled Dynamic Light Scattering Assay for Chemical and Biological Detection and Analysis July 2012 Copyright Nano Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954 Email: sales@nanodiscoveryinc.com

Part I. General introduction Part II. NanoDLSay™ for protein research Part III. Comparison with other analytical techniques Part IV. NDS1200 – the instrument for NanoDLSay™

Part I. General Introduction The principle of NanoDLSay™ How to conduct NanoDLSay™ Applications and examples Analytical performance and advantages

What is NanoDLSay™: Detect the target analytes by monitoring the size change of nanoparticles upon binding with the target analyte Gold nanoparticle (AuNP) AuNP immunoprobe D » 120 nm AuNP immunoprobe bound with a small protein monomer D » 130-160 nm AuNP immunoprobe bound with a large protein complex D > 130-160 nm Y AuNPs bound with metal ion targets through metal-chelating ligands AuNPs bound with small chemical targets through coordinative ligand interactions Unmodified AuNP D = 100 nm D >> 100 nm 2+ D >> 100-200 nm General assay format: AuNP clusters formed from binding with target analytes

Two assay formats I. Individual particle size increase Suitable for large analytes such as proteins, complexes and viruses Suitable for kinetic binding studies Y Y II. Nanoparticle cluster formation Suitable for any analytes, especially for chemicals and ions Provide best sensitivity

What is dynamic light scattering (DLS): Measure particle size in nanometer size range Scattered light intensity fluctuation Correlation function Laser beam Scattering light NDS1200 Instrument

Why Gold Nanoparticles (AuNPs)? Exceptionally intense light scattering property 105 times stronger than a fluorescent dye molecule; 100s 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). (C) A dark field optical image of gold nanorods (AuNR)

How to conduct NanoDLSay™? Step 1. Prepare the AuNP probe Step 2. Mix the AuNP probe with the sample solution Step 3. Incubate the assay solution Step 4. Measure the particle size of the assay solution Dose-response curve Target concentration Average particle size (nm) Unknown sample Standard curve A typical assay condition: Mix 40 µL AuNP probe with 2 µL sample Incubate 5-15 min at room temperature Analyze the particle size to obtain results Read-out: average particle size (nm)

AuNP Bioconjugate Preparation 1. Direct adsorption method Easy to use but lower stability; primary choice Citrate AuNPs (100 nm) (Ted Pella Inc.) (1 mL AuNP + 5-10 µg antibody) Blocking reagent: Bovine serum albumin (BSA) (2.5 mg/mL) Centrifuge Re-dispersion ~15 min ~30 min 2. Covalent conjugation method More complicated but higher stability NHS/EDC activation -NH2 or -COOH Centrifuge Re-dispersion Functional ligand-coated AuNPs

Applications of NanoDLSay™ Proteins DNAs RNAs Viruses Small chemicals Toxic metal ions

Examples Liu X, et al. 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. Chun C, et al. A facile and sensitive immunoassay for the detection of alpha- fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering. Chem. Comm. 2011, 47, 11047-11049.  Kalluri JR, 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. Gao D, et al. An ultrasensitive method for the detection of gene fragment from transgenics using label-free gold nanoparticle probe and dynamic light scattering. Anal. Chim Acta 2011; 696:1-5.  Driskell JD, et al. One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst 2011; 136:3083-3090. Wang X, et al. Detection of hepatitis B surface antigen by target-induced aggregation monitored by dynamic light scattering. Anal. Biochem. 2012, online. For a more complete list, visit: www.nanodiscoveryinc.com

Analytes Sensitivity Dynamic Range Analytical Performance Analytes Sensitivity Dynamic Range Proteins High pg/mL to low ng/mL range 2-3 orders of magnitude DNAs 30 fM (5 orders of magnitude more sensitive than SPR and fluorescence techniques) > 5 orders of magnitude Viruses < 100 TCID50/mL (1-2 orders of magnitude more sensitive than commercial diagnostic kits) Toxic metal ions Arsenics: 10 ppt (WHO acceptable limit: 10 ppb) Lead: 100 ppt (2 orders of magnitude below the EPA standard limit) Small molecules 7 nM (5 orders of magnitude more sensitive than the colorimetric method) > 4 orders of magnitude Explosive chemicals 100 pM Notes: (1) ng-nanogram; fg-femtogram; fM-femtomolar; pM-picomolar; nM-nanomolar; ppb-parts per billion; ppt-parts per trillion; TCID50- 50% tissue culture infective dose. (2) All data were taken from published papers. Refer to the list of publications for more information. (3) WHO: World Health Organization; EPA: Environmental Protection Agency.

Analytical Performance Comparison of NanoDLSay™ with other methods for DNA detection Label Method Detection limit AuNP Colorimetric 1 × 10-8 mol/L Au chip Surface plasmon resonance 1 × 10-9 mol/L Au/polyaniline nantube Electrochemical impedance spectroscopy 3 × 10-13 mol/L Quantum dots Anodic stripping voltammetry 5 × 10-11 mol/L ZnS and CdSe quantum dots Fluorescence 2 × 10-9 mol/L NanoDLSay™ Dynamic light scattering 3 × 10-14 mol/L Ref: Gao D, Sheng Z, Han H. An ultrasensitive method for the detection of gene fragment from transgenics using label-free gold nanoparticle probe and dynamic light scattering. Anal. Chim Acta 2011; 696:1-5.

The ultrahigh sensitivity of NanoDLSay™ AuNP monomer versus clusters Size 100 nm ~300 nm Scattered light intensity ratio 1 ~1000‡ Number (molar) ratio 99.9% (10 pM) 0.1% (10 fM) Net scattered light intensity Intensity-averaged particle size ½ * 100 nm + ½ * 300 nm = 200 nm ‡: Calculated according to Mie scattering theory From the above illustration, it can be seen that with a trace amount of AuNP cluster formation due to target analyte binding, the intensity-averaged particle size increases substantially - The origin of high sensitivity of NanoDLSay™

Advantages of NanoDLSay™ Requires a small volume of sample (1-5 µL) Obtain results in several minutes Single-step assay procedure Extremely simple and easy to use High to ultra-high sensitivity Excellent reproducibility Extremely low cost of consumables

Part II. NanoDLSay™ for Protein Research Introduction Protein detection and concentration analysis Kinetic study of protein-protein interaction Label-free protein complex detection and binding partner analysis Label-free protein oligomer/aggregate detection and analysis

X Introduction: Understand the problems of traditional immunoassay… B Individual protein monomer Protein complex Protein aggregates A protein does not stay alone in biological systems… A Traditional immunoassay assumes proteins exist alone antibody X B Traditional immunoassay likely fails to detect proteins in complexes

NanoDLSay™: Detect target proteins in all forms Unique capabilities Average particle size increase (nm) Incubation time (min)  = 2D of analyte 0 min 30 min 1 2 3 Kinetic binding study: monitor the particle size change continuously during the assay Determine the “size” of the target analyte at a saturated binding level Determine if a target protein is a monomer, complex, or aggregates Label-free detection: no need to label the target proteins Detection of protein complexes and aggregates from real biological samples

1. Protein detection and concentration analysis Dose-response curve Target concentration Average particle size (nm) Unknown sample Standard curve Y Single-probe assay Y Y Y Y Y Y Standard curve is established using standard solutions Relative quantitation can be done by directly comparing the average particle size Two-probe assay or single polyclonal antibody probe assay (higher sensitivity)

2. Kinetic study of protein-protein interaction Alternative: Target A Target B Procedure: Immobilize one target A protein to the AuNP Mix the target A-modified AuNP with target B protein Monitor the AuNP size change Binding affinity may be estimated using Langmuir adsorption model Average particle size (nm) Non-binding proteins Incubation time (min) 30

3. Label-free protein complex detection and binding partner analysis Average particle size increase (nm) Incubation time (min) Step 2: Binding partner screening using antibody Step 1: Catch the target Particle size change upon antibody addition c  ~ 2D Binding partners Not binding partners 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. Analyze the binding partners to the target protein

4. 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

References Protein-protein or other biomolecular interactions Jans H, et al. Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding study. Anal. Chem. 2009; 81: 9425-9432. Austin L, et al. An immunoassay for monoclonal antibody isotyping and quality analysis using gold nanoparticles and dynamic light scattering. American Biotechnology Laboratory 2010; 28: 8, 10-12. Sánchez-Pomales G, et al. A lectin-based gold nanoparticle assay for proving glycosylation of glycoproteins. Biotechnology Bioengineering 2012, published online. Wang, X.; Ramström, O.; Yan, M. Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions. Analyst 2011, 136, 4174-4178. Label-free protein complex detection and binding partner analysis Jaganathan S, et al. A functional nuclear epidermal growth factor receptor, Src and Stat3 heteromeric complex in pancreatic cancer cells. PLoS One 2011, 6(5):e19605. Label-free protein oligomer/aggregate detection Bogdanovic J, et al. A label-free nanoparticle aggregation assay for protein complex/aggregate detection and analysis. Anal. Biochem. 2010; 45:96-102. Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:259-265.

Part III. Comparison of NanoDLSay™ with other analytical techniques ELISA (enzyme-linked immunoabsorbent assay) Surface plasmon resonance Co-immunoprecipitation/immunoblotting Size exclusion chromatography Analytical Ultracentrifugation Colorimetric assay using AuNP probes

1. NanoDLSay™ versus ELISA Sandwich ELISA Likely fail to detect complexed proteins Results obtained in 2-3 hours Multiple steps – extensive labor Relatively large sample volume (10-100s µL) NanoDLSay™ Detect target protein in all forms Reveal more accurate biological information Reveal protein complex state Results obtained in several minutes Single step process Samll sample volume (1-5 µL)

2. 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

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

Non-specific interactions 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

4. 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 Not suitable for absolute quantitative analysis of various oligomers

5. Comparison of NanoDLSay™ with colorimetric assay Target analyte binding-induced AuNP cluster formation causes SPR band shift of AuNPs to longer wavelength - Color change Y Color change Colorimetric assay Easy to perform With or without instrument Low sensitivity Does not reveal molecular size information Not suitable for colored samples (e.g. blood) Wavelength (nm) Absorbance (A.U.) 400 800 Before assay After assay 600 500 700

Product & Services Part IV. 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 convenient, flexible 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