One New Technology, Discover a New World
NanoDLSay™: Nanoparticle-Enabled
Dynamic Light Scattering Assay for Chemical
and Biological Detection and Analysis
July 2012
Copyright Nano Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954
Email: sales@nanodiscoveryinc.com
Part I. General introduction
Part II. NanoDLSay™ for protein research
Part III. Comparison with other analytical techniques
Part IV. NDS1200 – the instrument for NanoDLSay™
Part I. General Introduction
o The principle of NanoDLSay™
o How to conduct NanoDLSay™
o Applications and examples
o Analytical performance and advantages
What is NanoDLSay™: Detect the target analytes by monitoring the
size change of nanoparticles upon binding with the target analyte
Gold nanoparticle (AuNP)
Y
Y
D = 100 nm
Unmodified AuNP
D » 120 nm
D » 130-160 nm
AuNP immunoprobe
AuNP immunoprobe
bound with a small
protein monomer
Y
D > 130-160 nm
AuNP immunoprobe
bound with a large
protein complex
2+
D >> 100-200 nm
2+
General assay format:
AuNP clusters formed from
binding with target analytes
2+
2+
AuNPs bound with metal ion targets
through metal-chelating ligands
D >> 100 nm
AuNPs bound with small chemical targets
through coordinative ligand interactions
Two assay formats
Y
I. Individual particle size increase
• Suitable for large analytes such as
proteins, complexes and viruses
• Suitable for kinetic binding studies
Y
Y
II. Nanoparticle cluster formation
Y
Y
• Suitable for any analytes,
especially for chemicals and ions
• Provide best sensitivity
What is dynamic light scattering (DLS):
Measure particle size in nanometer size range
Scattering light
Laser beam
Scattered light intensity fluctuation
Correlation function
NDS1200 Instrument
Why Gold Nanoparticles (AuNPs)?
•
•
•
•
•
Exceptionally intense light scattering property
105 times stronger than a fluorescent dye molecule;
100s 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). (C) A dark field optical image of gold nanorods (AuNR)
How to conduct NanoDLSay™?
• Step 1. Prepare the AuNP probe
• Step 2. Mix the AuNP probe with the sample solution
• Step 3. Incubate the assay solution
• Step 4. Measure the particle size of the assay solution
A typical assay condition:
1. Mix 40 µL AuNP probe with 2 µL sample
2. Incubate 5-15 min at room temperature
3. Analyze the particle size to obtain results
Read-out: average particle size (nm)
Average particle size (nm)
Dose-response curve
Unknown sample
Standard curve
Target concentration
AuNP Bioconjugate Preparation
1. Direct adsorption method
Easy to use but lower stability; primary choice
~15 min
Citrate AuNPs (100 nm)
(Ted Pella Inc.)
(1 mL AuNP + 5-10 µg antibody)
2. Covalent conjugation method
~30 min
Blocking reagent:
Bovine serum albumin
(BSA) (2.5 mg/mL)
1. Centrifuge
2. Re-dispersion
More complicated but higher stability
NHS/EDC activation
-NH2 or -COOH
Functional ligand-coated AuNPs
1. Centrifuge
2. Re-dispersion
Proteins
Viruses
Applications of
NanoDLSay™
Small
chemicals
DNAs
RNAs
Toxic
metal ions
Examples
Liu X, et al. A One-step homogeneous immunoassay for cancer biomarker
detection using gold nanoparticle probes coupled with dynamic light scattering. J.
Am. Chem. Soc. 2008; 130:2780-2782.
Chun C, et al. A facile and sensitive immunoassay for the detection of alphafetoprotein using gold-coated magnetic nanoparticle clusters and dynamic
light scattering. Chem. Comm. 2011, 47, 11047-11049. 
Kalluri JR, et al. Use of gold nanoparticles in a simple colorimetric and
ultrasensitive dynamic light scattering assay: selective detection of arsenic in
groundwater. Angew. Chem. Int. Ed. 2009; 48:9668-9671.
Gao D, et al. An ultrasensitive method for the detection of gene fragment from
transgenics using label-free gold nanoparticle probe and dynamic light scattering.
Anal. Chim Acta 2011; 696:1-5. 
Driskell JD, et al. One-step assay for detecting influenza virus using dynamic
light scattering and gold nanoparticles. Analyst 2011; 136:3083-3090.
Wang X, et al. Detection of hepatitis B surface antigen by target-induced
aggregation monitored by dynamic light scattering. Anal. Biochem. 2012, online.
For a more complete list, visit: www.nanodiscoveryinc.com
Analytes
Proteins
DNAs
Viruses
Analytical Performance
Sensitivity
High pg/mL to low ng/mL range
Dynamic Range
2-3 orders of magnitude
30 fM
> 5 orders of magnitude
(5 orders of magnitude more sensitive than SPR
and fluorescence techniques)
< 100 TCID50/mL
(1-2 orders of magnitude more sensitive than
commercial diagnostic kits)
2-3 orders of magnitude
Toxic metal ions
Arsenics: 10 ppt
(WHO acceptable limit: 10 ppb)
Lead: 100 ppt
(2 orders of magnitude below the EPA standard
limit)
2-3 orders of magnitude
Small molecules
7 nM
(5 orders of magnitude more sensitive than the
colorimetric method)
> 4 orders of magnitude
100 pM
2-3 orders of magnitude
Explosive chemicals
Notes:
(1) ng-nanogram; fg-femtogram; fM-femtomolar; pM-picomolar; nM-nanomolar; ppb-parts
per billion; ppt-parts per trillion; TCID50- 50% tissue culture infective dose. (2) All data were
taken from published papers. Refer to the list of publications for more information. (3) WHO:
World Health Organization; EPA: Environmental Protection Agency.
Analytical Performance
Comparison of NanoDLSay™ with other methods for DNA detection
Label
Method
Detection
limit
AuNP
Colorimetric
1 × 10-8 mol/L
Au chip
Surface plasmon resonance
1 × 10-9 mol/L
Au/polyaniline
nantube
Electrochemical impedance
spectroscopy
3 × 10-13 mol/L
Quantum dots
Anodic stripping voltammetry
5 × 10-11 mol/L
ZnS and CdSe
quantum dots
Fluorescence
2 × 10-9 mol/L
NanoDLSay™
Dynamic light scattering
3 × 10-14 mol/L
Ref: Gao D, Sheng Z, Han H. An ultrasensitive method for the detection of gene fragment from
transgenics using label-free gold nanoparticle probe and dynamic light scattering. Anal. Chim Acta
2011; 696:1-5.
The ultrahigh sensitivity of NanoDLSay™
AuNP monomer versus
clusters
Size
100 nm
~300 nm
Scattered light intensity ratio
1
~1000‡
Number (molar) ratio
99.9% (10 pM)
0.1% (10 fM)
Net scattered light intensity
1
1
Intensity-averaged particle size
‡:
½ * 100 nm + ½ * 300 nm = 200 nm
Calculated according to Mie scattering theory
From the above illustration, it can be seen that with a trace amount of AuNP
cluster formation due to target analyte binding, the intensity-averaged particle
size increases substantially - The origin of high sensitivity of NanoDLSay™
Advantages of NanoDLSay™
o Requires a small volume of sample (1-5 µL)
o Obtain results in several minutes
o Single-step assay procedure
o Extremely simple and easy to use
o High to ultra-high sensitivity
o Excellent reproducibility
o Extremely low cost of consumables
Part II. NanoDLSay™ for Protein Research
o Introduction
o Protein detection and concentration analysis
o Kinetic study of protein-protein interaction
o Label-free protein complex detection and
binding partner analysis
o Label-free protein oligomer/aggregate
detection and analysis
Introduction: Understand the problems of
traditional immunoassay…
A protein does not stay alone in biological systems…
Individual protein
monomer
A
Protein complex
antibody
Protein aggregates
B
X
Traditional immunoassay
assumes proteins exist alone
Traditional immunoassay likely fails
to detect proteins in complexes
NanoDLSay™: Detect target proteins in all forms
Unique capabilities
3
Average particle size increase (nm)


2


1
 = 2D of analyte
0 min
Incubation time (min)
30 min

Kinetic binding study:
monitor the particle size
change continuously during
the assay
Determine the “size” of the
target analyte at a saturated
binding level
Determine if a target protein
is a monomer, complex, or
aggregates
Label-free detection: no
need to label the target
proteins
Detection of protein
complexes and aggregates
from real biological samples
1. Protein detection and concentration analysis
Dose-response curve
Average particle size (nm)
Y
Y
• Single-probe assay
Y
Y
Y
Unknown sample
Standard curve
Target concentration
Y
Y
Y
• Two-probe assay or single polyclonal
antibody probe assay (higher sensitivity)
o Standard curve is established
using standard solutions
o Relative quantitation can be
done by directly comparing the
average particle size
2. Kinetic study of protein-protein interaction
Alternative:
Target A
Average particle size (nm)
Target B
Non-binding proteins
0
Incubation time (min)
30
Procedure:
1. Immobilize one target A
protein to the AuNP
2. Mix the target A-modified
AuNP with target B protein
3. Monitor the AuNP size change
4. Binding affinity may be
estimated using Langmuir
adsorption model
Average particle size increase (nm)
3. Label-free protein complex detection and
binding partner analysis
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. Analyze the binding partners to the target protein
4. Label-free protein oligomer/aggregate
detection and analysis
protein monomer
• Specific detection of target protein
oligomer/aggregates in real samples
Average particle size increase (nm)
• Protein oligomer/aggregates cause
AuNP probe cluster formation
oligomers,
aggregates
 = 2D of analyte
0 min
Incubation time (min)
30 min
References
Protein-protein or other biomolecular interactions
Jans H, et al. Dynamic light scattering as a powerful tool for gold nanoparticle
bioconjugation and biomolecular binding study. Anal. Chem. 2009; 81: 9425-9432.
Austin L, et al. An immunoassay for monoclonal antibody isotyping and quality analysis
using gold nanoparticles and dynamic light scattering. American Biotechnology
Laboratory 2010; 28: 8, 10-12.
Sánchez-Pomales G, et al. A lectin-based gold nanoparticle assay for proving
glycosylation of glycoproteins. Biotechnology Bioengineering 2012, published online.
Wang, X.; Ramström, O.; Yan, M. Dynamic light scattering as an efficient tool to study
glyconanoparticle-lectin interactions. Analyst 2011, 136, 4174-4178.
Label-free protein complex detection and binding partner analysis
Jaganathan S, et al. A functional nuclear epidermal growth factor receptor, Src and
Stat3 heteromeric complex in pancreatic cancer cells. PLoS One 2011, 6(5):e19605.
Label-free protein oligomer/aggregate detection
Bogdanovic J, et al. A label-free nanoparticle aggregation assay for protein
complex/aggregate detection and analysis. Anal. Biochem. 2010; 45:96-102.
Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a
nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:259-265.
Part III. Comparison of NanoDLSay™ with
other analytical techniques
o ELISA (enzyme-linked immunoabsorbent assay)
o Surface plasmon resonance
o Co-immunoprecipitation/immunoblotting
o Size exclusion chromatography
o Analytical Ultracentrifugation
o Colorimetric assay using AuNP probes
1. NanoDLSay™ versus ELISA
Sandwich ELISA
o Likely fail to detect complexed proteins
o Results obtained in 2-3 hours
o Multiple steps – extensive labor
o Relatively large sample volume (10-100s µL)
NanoDLSay™
o Detect target protein in all forms
o Reveal more accurate biological information
o Reveal protein complex state
o Results obtained in several minutes
o Single step process
o Samll sample volume (1-5 µL)
2. 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
3. Comparison of NanoDLSay™ with co-immunoprecipitation
(Co-IP) followed by immunoblotting for protein complex analysis
Non-specific interactions
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
4. 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
o Not suitable for absolute quantitative analysis of various oligomers
5. Comparison of NanoDLSay™ with colorimetric assay
Y
Y
Y
Y
Color change
Target analyte binding-induced
AuNP cluster formation causes
SPR band shift of AuNPs to
longer wavelength
- Color change
Absorbance (A.U.)
Colorimetric assay
Before assay
o Easy to perform
o With or without instrument
o Low sensitivity
After assay
o Does not reveal molecular
size information
400
500
600
Wavelength (nm)
700
800
o Not suitable for colored
samples (e.g. blood)
Product & Services
Part IV.
NDS1200: A new dynamic light scattering instrument
designed for performing NanoDLSay™

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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
convenient, flexible 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