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NanoDLSay™: A Most Comprehensive Tool for Protein Detection and Analysis Nano Discovery Inc. Copyright Nano Discovery Inc. www.nanodiscoveryinc.com NANO

Nano Discovery Inc. Copyright Nano Discovery Inc. NANO

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NanoDLSay™: A Most Comprehensive Tool for Protein Detection and

Analysis

Nano Discovery Inc.

Copyright Nano Discovery Inc.www.nanodiscoveryinc.com

NANO

What is NanoDLSay™?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!

How to conduct NanoDLSay™?

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 assa

y

Inte

nsi

ty d

istr

ibuti

on

Avera

ge p

art

icle

si

ze

Protein concentration

Measure by DLS Extract analyte information

Add sample

100 nm 150 nm

All you need to do is to add the sample to the AuNP probe solution! !!

NDS-1200: The Instrument for NanoDLSay™

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

Application NotesAll following applications were based on reported, peer-reviewed scientific

publications

I. Protein-Protein Interaction and Binding Study

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.

Avera

ge p

art

icle

siz

e incr

ease

(n

m)

Protein concentration or incubation time

target proteinbinding partner of the target protein

+

Association and dissociation rate constant, ka and kd, can be determined. Method described in the NDS-1200 User Manual

Comparison of NanoDLSay™ with Surface Plasmon Resonance (SPR)

Technique for Protein-Protein Interaction Study

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

II. Antibody Isotyping and Quality Control

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.

Avera

ge p

art

icle

siz

e incr

ease

(n

m)

Protein concentration or incubation time

Matched antibody isotype

Anti-isotype AuNP probe

Unmatched antibody isotype

Minimum size increase

Substantial size increase

III. Protein Complex Detection and Binding Partner Analysis

Avera

ge p

art

icle

siz

e incr

ease

(n

m)

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).

Continued: Binding Partner Analysis

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).

c

Avera

ge p

art

icle

siz

e incr

ease

(n

m)

Incubation time

Antibody screening

c

Particle size increase following antibody addition to the assay solution

Conclusion:Protein and are binding partners of

Comparison of NanoDLSay™ with Co-Immunoprecipitation Followed by

Immunoblotting for Protein Complex Binding Partner Analysis

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

IV. Label-free Protein Aggregate Detection

Particle size distribution curve (nm)

Protein monomer and small complex:Smaller nanoparticle size increaseMonodispersed particle size distribution

Inte

nsi

ty

dis

trib

uti

on

Protein aggregates:Substantially larger nanoparticle size increaseMuch 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.

Why NanoDLSay™ is unique in protein aggregate detection and study?

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

It is a label-free technique to detect protein aggregates directly from un-purified biological samples and fluidsIt enables the detection of protein aggregates at much higher sensitivity

NanoDLSay™ enables researchers to discover new molecular information associated with human

diseases that have not been revealed using other existing techniques

Contact Information

NANO

Nano Discovery Inc. 12565 Research Parkway Suite 400Orlando, FL 32826

www.nanodiscoveryinc.comTel: 407-770-8954Email: [email protected]

Copyright Nano Discovery Inc. June 2011

Disclaimer:1. Patent application pending on NanoDLSay™ technology: PCT/US09/030087 and PCT/US11/210022. Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay™ technology