1. 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 Tel:

2. 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.

3. 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 ~ nm
D >> nm

4. 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)

5. 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,
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,
3. Calzolai L, Franchini F, Gilliland D, Rossi F, Protein—nanoparticle interaction: identification of the ubiquitin-gold nanoparticle interaction site. Nano Lett ; 10:

6. 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

7. 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)

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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:

14. 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,
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

15. 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

16. 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 nm, 70 nm of increase suggests it is a complex!
~ 70 nm
Control study

17. 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

18. 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

19. 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:

20. 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

21. Comparison of NanoDLSay™ with other existing techniques

22. 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

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

24. 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

25. 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

26. 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: samples/hour
The hardware is maintenance-free
No special housing environment is required for the instrument
Extremely easy-to-use software

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

28. 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:
Or visit online:
www. nanodiscoveryinc.com
Notes
Patent application pending on NanoDLSay™ technology and NDS1200 system: PCT/US09/ and PCT/US11/21002
Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay™ technology