Transcript NanoDLSay™: A Most Comprehensive Tool for Protein

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Nano Discovery Inc.
Copyright Nano Discovery Inc.
www.nanodiscoveryinc.com
NANO
Nanoparticle-Enabled Dynamic Light Scattering Assay
~100 nm
~120 nm
Gold Nanoparticle
(AuNP)
AuNP-antibody
immunoprobe
~130-150 nm
Immunoprobe bound
with target protein
Target protein is detected by monitoring the nanoparticle size change!
>>130-150 nm
Immunoprobe bound with
target protein complex
Step I. Add sample to
the nanoparticle
probe solution
Step III. Measure the
particle size change
of the assay solution
Step II. Incubate the
assay solution
100 nm
150 nm
Before
assay
After
assay
Average particle size (nm)
Measure by DLS
Average particle size
Intensity distribution
Add sample
Protein concentration
Extract analyte information
All you need to do is to add the sample to the AuNP probe solution! !!
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
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All following applications were based on reported, peer-reviewed scientific publications
Average particle size increase (nm)
Association and dissociation rate constant,
ka and kd, can be determined. Method
described in the NDS-1200 User Manual
target protein
+
binding partner of the target protein
Protein concentration or incubation time
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.
NanoDLSay™
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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
Add sample
SPR
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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
Sample solution flow through
Homogeneous
solution assay
SPR substrate: gold monolayer film
Average particle size increase (nm)
Substantial size increase
Anti-isotype AuNP probe
Matched antibody isotype
Unmatched antibody isotype
Minimum size increase
Protein concentration or incubation time
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)
A size increase substantially
larger than 2D indicates the
presence of protein complex
Maximum size increase
 2 D (diameter of protein
)
Incubation time
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).
Average particle size increase (nm)
Antibody screening
c
Particle size increase
following antibody addition
to the assay solution
Conclusion:
Protein
and
are binding partners of
c
Incubation time
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).
NanoDLSay™
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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
Step1.
Catch the target
Step2.
Analyze the binding partner
Co-immunoprecipitation
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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
Step1.
Catch
Step 2.
Centrifuge/Isolate
Multiple-steps:
Immunoblotting
No isolation between step 1 and 2
Step 3.
Detach protein
from the beads
Protein aggregates:
Substantially larger nanoparticle size increase
Much broader particle size distribution curve
Intensity distribution
Protein monomer and small complex:
Smaller nanoparticle size increase
Monodispersed particle size distribution
Particle size distribution curve (nm)
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.
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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
Nano Discovery Inc.
12565 Research Parkway Suite 400
Orlando, FL 32826
www.nanodiscoveryinc.com
Tel: 407-770-8954
Email: [email protected]
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
NANO
Copyright Nano Discovery Inc.
June 2011