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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. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954
Email: huo@nanodiscoveryinc.com
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
I.
Protein complexes
Protein aggregates
II.
Traditional immunoassay assumes
proteins only exist as monomer!
III.
•
•
We will not know if any complex is here or not
The complex leads to a fake lower concentration
The labeled signaling antibody cannot
recognize the complexed target protein
Conclusion in this case: target protein
is not present in the sample at all!!!
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 ~ 120 nm
D ~ 130-160 nm
D >> 130-160 nm
D = 100 nm
Gold nanoparticles
(AuNP)
Dynamic light scattering (DLS):
Measure particle size in nanometer size range
Scattered light intensity fluctuation
Scattering light
Laser beam
Intensity Distr. (%)
Correlation function
Large particle
Small particle
100
120
200
Average particle size (nm)
Protein-AuNP interactions: Au-S, Au-N bonding,
electrostatic and van der Waals interaction
Protein
“Protein Corona”
References
Au0
Au+
Au+
Au0
Au0
Au+
Au+
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.
Protein
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.
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
B
A
C
PS particle
Serum
AuNPs
Gold nanorods
AuNPs
Dark field optical images of AuNPs mixed with human serum (A) and PS
particles (B) and gold nanorod AuNR (C)
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
Average particle size increase (nm)
3
2
1
 = 2D of analyte
0 min
Incubation time (min)
30 min
Information on the target molecule is
obtained from real biological samples
Applications
 Real-time kinetic binding study of proteinprotein 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
Assay format I: immobilize one target protein on AuNP as a probe
Procedure:
1. Immobilize one target A or B
protein to the AuNP
2. Mix the target A or B-modified
AuNP with target B or A protein
3. Monitor the AuNP size change
4. Binding affinity may be estimated
using Langmuir adsorption model
Target A
Average particle size (nm)
Target B
Requirements:
Immobilization of A or B on AuNP does
not affect protein-protein interactions
Non-binding proteins
0
Incubation time (min)



30
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
Assay format II: allow proteins bind first in solution, then conduct adsorption assay
+
A
+
B
C
complex
Comparison to obtain
binding information
Procedure:
1. Mix relevant protein binding partners together
2. Conduct adsorption assay of individual target with AuNP
3. Conduct adsorption assay of mixed product with AuNP
4. 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
I. Kinetic study of protein-protein interaction
Assay format III: two AuNP probe interaction assay
Target B
Target A
+
Procedure:
1. Make a target A or B-conjugated AuNP probe
2. Mix the two probes in solution
3. Monitor the size change of the assay
4. A-B interaction leads to AuNP cluster formation
Limitations:
Immobilization of A or B on AuNP does
not affect protein-protein interactions
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
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.
Kinetic binding study between protein Aconjugated AuNP with human IgG (target)
Binding activity of protein A-conjugated AuNP
with human IgG is lost after crosslinking
modification with glutaraldehyde (GA)
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.
mouse anti- f-PSA-B
mouse anti-f-PSA-A
120.0
Particle Size (nm)
Particle Size (nm)
145.0
anti-IgG1
135.0
anti-IgG2a
anti-IgG2b
125.0
anti-IgM
115.0
105.0
115.0
anti-IgG1
anti-IgG2a
110.0
anti-IgG2b
anti-IgM
105.0
0
5
10
15
Incubation Time (min)
20
25
0
5
10
15
20
Incubation Time (min)
25
30
Assay: conjugate anti-isotype antibody to AuNP, and then conduct assay on the antibody product
Product 1: Mouse anti-f-PSA antibody A, isotype IgG1, pure monoclonal antibody
Product 2: Mouse anti-f-PSA antibody B, mixture of IgG1, IgG2a and IgG2b
Both products were claimed to be mouse monoclonal antibodies



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
Average particle size increase (nm)
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
Binding partners
Particle size change
upon antibody addition
c
 ~ 2D
Not binding partners
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
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:
1. Prepare the AuNP immunoprobe for EGFR
2. Use the EGFR-AuNP probe to catch the target
3. Determine if EGFR is in a complex
4. Control: add anti-EGFR to the sample solution,
incubate, and then repeat the binding assay with
AuNP immunoprobe.
AuNP immunoprobe for
target protein, EGFR
160
~ 70 nm
120
140
Particle size (nm)
140
Particle size (nm)
EGFR is about 10-15 160
nm, 70 nm of
increase suggests it is a complex!
EGFR-AuNP probe
Mouse IgG1-AuNP probe
100
80
60
120
Control study
100
Untreated sample
80
Inhibited for 7 min
Inhibited for 24 min
60
40
0
10
20
30
Incubation time (min)
40
40
0
5
10
Incubation time (min)
15
Screening the binding partners in the complex using specific antibody
Step 2: Binding partner
screening using antibody
Experiments:
1. Add an antibody for the suspected binding partner
into the assay solution
2. If the particle size is increased, then it is a positive
response, and vice versa
3. Control: add a negative isotype control antibody to
the assay solution
40
39
34
Particle size increase (nm)
Particle size increase (nm)
35
30
29
If we conduct
a sandwich assay, the
conclusion
24 will be: EGFR is not there!
25
20
15
10
5
0
IgG
anti-STAT3
anti-SRC
anti-EGFR
19
Negative control
14
9
4
-1
IgG
anti-STAT3
anti-SRC
anti-EGFR
III. Label-free protein oligomer/aggregate detection and analysis
protein monomer
o Specific detection of target protein
oligomer/aggregates in real samples
Average particle size increase (nm)
o Protein oligomer/aggregates cause
AuNP probe cluster formation
oligomers,
aggregates
 = 2D of analyte
0 min
Incubation time (min)
30 min
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.
75
300
GAPDH in PB Buffer
Average particle size (nm)
Average particle size (nm)
GAPDH in TEA Buffer
250
200
150
Day I
Day II
100
Aggregation
begins here
50
0
65
60
55
50
45
0
20
40
60
80
100
GAPDH concentration (µg/mL)
120
80
0
20
40
60
80
100
GAPDH concentration (µg/mL)
120
GAPDH: Glyceraldehyde 3-Phosphate Dehydrogenase
GAPDH in RIPA Buffer
Average particle size (nm)
70
75
70
65
60
55
50
45
0
20
40
60
80
100
GAPDH concentration (µg/mL)
120
Experiments and results:
1. Conjugate an anti-GAPDH antibody to AuNP
2. Study the binding of anti-GAPDH-AuNP
probe with GAPDH at different concentrations
3. Plot an assay average particle size-GAPDH
concentration curve
4. Substantial aggregation of GAPDH occurs at
25 µg/mL in TEA buffer
5. Less aggregation in phosphate buffer
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).
IgG
Figure 2
5
10
100
huIgG concentration (µg/mL)
1000
tissue type
3
1
ra
de
0
G
60
Tumor-IgG interaction reflected in
the IgG-AuNP adsorption assay
2
68.5 nm
ra
de
80
85.5 nm
G
100
1
120
ra
de
140
NanoDLSay reveals
human IgG dimerization
at > 100 µg/mL
G
160
'Normal' threshold
set as 2SD from mean
be
ni
gn
180
corona size
Average particle size (nm)
200
300
290
280
270
260
250
240
230
220
210
200
190
180
170
160
150
140
130
120
110
100
al
IgG dimer
Average D ~ 300 nm
no
rm
Citrate-AuNP
D ~ 100 nm
Comparison of NanoDLSay™ with
other existing techniques
NanoDLSay™ versus Surface Plasmon Resonance (SPR)
NanoDLSay™
SPR
o Label-free technique
o Label-free technique
o Optical substrate: gold nanoparticle
o Optical substrate: gold thin film
o Read-out: AuNP size change
o Read-out: refractive index change
o Homogeneous solution assay
o Heterogeneous chip assay
o Low cost of consumables
o High cost of consumables
o Reveal the size information of the
target analyte, distinguish protein
complexes and oligomers/complexes
from monomers
o Does not reveal the size
information of the target analyte,
does not tell whether a protein is
a monomer, complex or oligomer
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
Product & Services
NDS1200: A new dynamic light scattering instrument
designed for performing NanoDLSay™
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
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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: 120180 samples/hour
The hardware is maintenance-free
No special housing environment is
required for the instrument
Extremely easy-to-use software
Product & Services
NanoDLSay™ software: A software designed for
flexible, kinetic and high throughput analysis
Order Information
Product and Order Information
NDS1200
NDS-Kit1000
Dynamic light scattering instrument for conducting NanoDLSay™
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: 407-770-8954
Email: sales@nanodiscoveryinc.com
Or visit online:
www. nanodiscoveryinc.com
Notes
o 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