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Copyright Nano Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954 Email: [email protected]
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
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.
A fundamental problem with the current bioassay
Individual proteins Protein complexes Protein aggregates
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.
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
What is NanoDLSay™: Detect target proteins by monitoring the size change of nanoparticles upon binding with the target protein
Gold nanoparticles (AuNP)
D = 100 nm
Protein monomer (~ 5-20 nm)
Protein complex (>> 5-20 nm)
Immunoglobulin G (IgG) (~ 7-10 nm)
D ~ 120 nm D ~ 130-160 nm D >> 130-160 nm
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
Average particle size (nm)
Inte
nsi
ty D
istr
. (%
)
100 120 200
Protein-AuNP interactions: Au-S, Au-N bonding, electrostatic and van der Waals interaction
Au0
Au+
Au0
Au0
Au+Au+
Au+
Protein
Protein
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, 106-117.
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, 365-379.
3. Calzolai L, Franchini F, Gilliland D, Rossi F, Protein—nanoparticle interaction: identification of the ubiquitin-gold nanoparticle interaction site. Nano Lett. 2010; 10: 3101-3105.
“Protein Corona”
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
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
AuNPs
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)
Ave
rage
par
ticle
siz
e in
crea
se (
nm)
Incubation time (min)
= 2D of analyte
0 min 30 min
1
2
3 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
NanoDLSay™: Detect target proteins in all forms
Information on the target molecule is obtained from real biological
samples
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
I. Kinetic study of protein-protein interaction
Procedure:1. Immobilize one target A or B
protein to the AuNP2. Mix the target A or B-modified
AuNP with target B or A protein3. Monitor the AuNP size change4. Binding affinity may be estimated
using Langmuir adsorption model
Target A
Target B
Ave
rage
par
ticle
siz
e (n
m)
Incubation time (min)0 30
Non-binding proteins
Assay format I: immobilize one target protein on AuNP as a probe
Requirements:Immobilization of A or B on AuNP does not affect protein-protein interactions
Sample suitability:Pure protein samples
Homogeneous solution assay, obtain results in minutes Detect both strong and weak binding Monitoring binding in real-time
I. Kinetic study of protein-protein interaction
Procedure:1. Mix relevant protein binding partners together2. Conduct adsorption assay of individual target with AuNP3. Conduct adsorption assay of mixed product with AuNP4. Comparison of assay results from 2 and 3 to obtain
complex information
Assay format II: allow proteins bind first in solution, then conduct adsorption assay
+
Comparison to obtain binding information
Requirements:At least one protein will readily adsorb to AuNP
Sample suitability:Pure protein samples
The AuNP-adsorption assay does not affect target protein binding Suitable for studying multi-binding partner (more than 2) complexes
A B complex
Compared to assay format I:
+C
I. Kinetic study of protein-protein interaction
Procedure:1. Make a target A or B-conjugated AuNP probe2. Mix the two probes in solution3. Monitor the size change of the assay4. A-B interaction leads to AuNP cluster formation
Assay format III: two AuNP probe interaction assay
Sample suitability:Pure protein samples
Increase assay sensitivity for detecting weak interactions Not limited to the size of the proteins or other target molecules
Target A Target B
+
Limitations:Immobilization of A or B on AuNP does not affect protein-protein interactions
Compared to assay format I and II:
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.
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.
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
II. Label-free protein complex detection and binding partner analysis from real samples
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
Ave
rage
par
ticle
siz
e in
crea
se (
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
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).
0 5 10 15 20 25 30 3540
60
80
100
120
140
160
EGFR-AuNP probe
Mouse IgG1-AuNP probe
Incubation time (min)
Pa
rtic
le s
ize
(n
m)
0 2 4 6 8 10 1240
60
80
100
120
140
160
Untreated sample
Inhibited for 7 min
Inhibited for 24 min
Incubation time (min)
Pa
rtic
le s
ize
(n
m)
Experiments:1. Prepare the AuNP immunoprobe for EGFR2. Use the EGFR-AuNP probe to catch the target3. Determine if EGFR is in a complex4. Control: add anti-EGFR to the sample solution,
incubate, and then repeat the binding assay with AuNP immunoprobe.
~ 70 nmEGFR is about 10-15 nm, 70 nm of increase suggests it is a complex!
Step 1: Catch the target
AuNP immunoprobe for target protein, EGFR
Control study
Experiments:1. Add an antibody for the suspected binding partner
into the assay solution2. If the particle size is increased, then it is a
positive response, and vice versa3. Control: add a negative isotype control antibody
to the assay solution
IgG anti-STAT3 anti-SRC anti-EGFR0
5
10
15
20
25
30
35
40
Par
ticl
e si
ze in
crea
se (
nm
)
IgG anti-STAT3 anti-SRC anti-EGFR-1
4
9
14
19
24
29
34
39
Par
ticl
e si
ze in
crea
se (
nm
)
If we conduct a sandwich assay, the conclusion will be: EGFR is not there!
Screening the binding partners in the complex using specific antibody
Step 2: Binding partner screening using antibody
Negative control
III. Label-free protein oligomer/aggregate detection and analysis
Ave
rage
par
ticle
siz
e in
crea
se (
nm)
Incubation time (min)
= 2D of analyte
0 min 30 min
protein monomer
oligomers,aggregates
o Protein oligomer/aggregates cause AuNP probe cluster formation
o Specific detection of target protein oligomer/aggregates in real samples
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.
Figure 2
tissue type
coro
na
size
normal
benig
n
Grade
1
Grade
2
Grade
3100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
'Normal' thresholdset as 2SD from mean
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-AuNPD ~ 100 nmIgG dimer
IgG
Average D ~ 300 nm
0 1 5 10 100 100060
80
100
120
140
160
180
200
68.5 nm
85.5 nm
huIgG concentration (µg/mL)
Ave
rage
par
ticle
siz
e (n
m)
NanoDLSay reveals human IgG dimerization at > 100 µg/mL
Tumor-IgG interaction reflected in the IgG-AuNP adsorption assay
Comparison of NanoDLSay™ with other existing techniques
NanoDLSay™ versus Surface Plasmon Resonance (SPR)
o Label-free technique
o Optical substrate: gold nanoparticle
o Read-out: AuNP size change
o Homogeneous solution assay
o Low cost of consumables
o Reveal the size information of the target analyte, distinguish protein complexes and oligomers/complexes from monomers
o Label-free technique
o Optical substrate: gold thin film
o Read-out: refractive index change
o Heterogeneous chip assay
o High cost of consumables
o Does not reveal the size information of the target analyte, does not tell whether a protein is a monomer, complex or oligomer
NanoDLSay™ SPR
Comparison of NanoDLSay™ with co-immunoprecipitation (Co-IP) followed by immunoblotting for protein complex analysis
NanoDLSay™ versus size exclusion chromatography (SEC) and analytical ultra-centrifugation (AU) for protein complex and oligomer/aggregate detection and analysis
SEC and AU:o For pure protein solution study only
o SEC underestimates complex or oligomer/aggregate formation
(eluent dilution disrupts existing complexes/oligomers)
o AU overestimates complex or oligomer/aggregate formation
(centrifugation artificially increases protein complexes/oligomers)
NanoDLSay™:o Detect protein complexes, oligomers/aggregates from real samples
o Fast screening test for protein complex/oligomer/aggregates
Non-specific interactions: effect on Co-IP and NanoDLSay™
A problem in Co-IP:
o Significant non-specific interactions caused by the separation process
o The concentration of the particle probes and proteins is artificially increased during centrifugation, increasing non-specific interactions
This problem does not exist in NanoDLSay™:
o The AuNP probe concentration is relatively low, reducing non-specific interactions
o No centrifugation separation is involved
NDS1200: A new dynamic light scattering instrument designed for performing NanoDLSay™
Product & Services
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 & ServicesNanoDLSay™ software: A software designed for flexible, kinetic and high throughput analysis
Noteso Patent application pending on NanoDLSay™ technology and NDS1200 system: PCT/US09/030087 and PCT/US11/21002 o Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay™ technology
Please Contact Us to Request a Quote:
NDS1200 Dynamic light scattering instrument for conducting NanoDLSay™
Assay kit including disposable sample cells and other consumables
3251 Progress Drive Suite A1Orlando, FL 32826Phone: 407-770-8954Email: [email protected]
NDS-Kit1000
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Or visit online: www. nanodiscoveryinc.com
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