Nano Discovery Inc. Copyright Nano Discovery Inc. NANO.
-
Upload
christine-gregory -
Category
Documents
-
view
253 -
download
1
Transcript of Nano Discovery Inc. Copyright Nano Discovery Inc. NANO.
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