1. 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 Tel:

2. Part I. General introduction
Part II. NanoDLSay™ for protein research
Part III. Comparison with other analytical techniques
Part IV. NDS1200 – the instrument for NanoDLSay™

3. Part I. General Introduction
The principle of NanoDLSay™
How to conduct NanoDLSay™
Applications and examples
Analytical performance and advantages

4. What is NanoDLSay™: Detect the target analytes by monitoring the size change of nanoparticles upon binding with the target analyte
Gold nanoparticle (AuNP)
AuNP immunoprobe
D » 120 nm
AuNP immunoprobe bound with a small protein monomer
D » nm
AuNP immunoprobe bound with a large protein complex
D > nm Y
AuNPs bound with metal ion targets through metal-chelating ligands
AuNPs bound with small chemical targets through coordinative ligand interactions
Unmodified AuNP
D = 100 nm
D >> 100 nm 2+
D >> nm
General assay format: AuNP clusters formed from binding with target analytes

5. Two assay formats 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
Suitable for any analytes, especially for chemicals and ions
Provide best sensitivity

6. What is dynamic light scattering (DLS): Measure particle size in nanometer size range
Scattered light intensity fluctuation
Correlation function
Laser beam
Scattering light
NDS1200 Instrument

7. 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
AuNPs
Serum A
PS particle B C
Gold nanorods
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)

8. 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
Dose-response curve
Target concentration
Average particle size (nm)
Unknown sample
Standard curve
A typical assay condition:
Mix 40 µL AuNP probe with 2 µL sample
Incubate 5-15 min at room temperature
Analyze the particle size to obtain results
Read-out: average particle size (nm)

9. AuNP Bioconjugate Preparation
1. Direct adsorption method
Easy to use but lower stability; primary choice
Citrate AuNPs (100 nm)
(Ted Pella Inc.)
(1 mL AuNP µg antibody)
Blocking reagent:
Bovine serum albumin (BSA) (2.5 mg/mL)
Centrifuge
Re-dispersion
~15 min
~30 min
2. Covalent conjugation method
More complicated but higher stability
NHS/EDC activation
-NH2 or -COOH
Centrifuge
Re-dispersion
Functional ligand-coated AuNPs

10. Applications of NanoDLSay™
Proteins
DNAs
RNAs
Viruses
Small
chemicals
Toxic
metal ions

11. 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:
Chun C, et al. A facile and sensitive immunoassay for the detection of alpha- fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering. Chem. Comm. 2011, 47, 
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:
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:
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:

12. Analytes Sensitivity Dynamic Range
Analytical Performance
Analytes
Sensitivity
Dynamic Range
Proteins
High pg/mL to low ng/mL range
2-3 orders of magnitude
DNAs
30 fM
(5 orders of magnitude more sensitive than SPR and fluorescence techniques)
> 5 orders of magnitude
Viruses
< 100 TCID50/mL
(1-2 orders of magnitude more sensitive than commercial diagnostic kits)
Toxic metal ions
Arsenics: 10 ppt
(WHO acceptable limit: 10 ppb)
Lead: 100 ppt
(2 orders of magnitude below the EPA standard limit)
Small molecules
7 nM
(5 orders of magnitude more sensitive than the colorimetric method)
> 4 orders of magnitude
Explosive chemicals
100 pM
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.

13. 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 × mol/L
Quantum dots
Anodic stripping voltammetry
5 × mol/L
ZnS and CdSe quantum dots
Fluorescence
2 × 10-9 mol/L
NanoDLSay™
Dynamic light scattering
3 × 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.

14. 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
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™

15. Advantages of NanoDLSay™
Requires a small volume of sample (1-5 µL)
Obtain results in several minutes
Single-step assay procedure
Extremely simple and easy to use
High to ultra-high sensitivity
Excellent reproducibility
Extremely low cost of consumables

16. Part II. NanoDLSay™ for Protein Research
Introduction
Protein detection and concentration analysis
Kinetic study of protein-protein interaction
Label-free protein complex detection and binding partner analysis
Label-free protein oligomer/aggregate detection and analysis

17. X Introduction: Understand the problems of traditional immunoassay… B
Individual protein monomer
Protein complex
Protein aggregates
A protein does not stay alone in biological systems… A
Traditional immunoassay assumes proteins exist alone
antibody X B
Traditional immunoassay likely fails to detect proteins in complexes

18. NanoDLSay™: Detect target proteins in all forms
Unique capabilities
Average particle size increase (nm)
Incubation time (min)
 = 2D of analyte
0 min
30 min 1 2 3
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

19. 1. Protein detection and concentration analysis
Dose-response curve
Target concentration
Average particle size (nm)
Unknown sample
Standard curve Y
Single-probe assay Y Y Y Y Y Y
Standard curve is established using standard solutions
Relative quantitation can be done by directly comparing the average particle size
Two-probe assay or single polyclonal antibody probe assay (higher sensitivity)

20. 2. Kinetic study of protein-protein interaction
Alternative:
Target A
Target B
Procedure:
Immobilize one target A protein to the AuNP
Mix the target A-modified AuNP with target B protein
Monitor the AuNP size change
Binding affinity may be estimated using Langmuir adsorption model
Average particle size (nm)
Non-binding proteins
Incubation time (min) 30

21. 3. Label-free protein complex detection and binding partner analysis
Average particle size increase (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
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

22. 4. 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

23. 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:
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,
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,
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:
Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:

24. Part III. Comparison of NanoDLSay™ with other analytical techniques
ELISA (enzyme-linked immunoabsorbent assay)
Surface plasmon resonance
Co-immunoprecipitation/immunoblotting
Size exclusion chromatography
Analytical Ultracentrifugation
Colorimetric assay using AuNP probes

25. 1. NanoDLSay™ versus ELISA
Sandwich ELISA
Likely fail to detect complexed proteins
Results obtained in 2-3 hours
Multiple steps – extensive labor
Relatively large sample volume (10-100s µL)
NanoDLSay™
Detect target protein in all forms
Reveal more accurate biological information
Reveal protein complex state
Results obtained in several minutes
Single step process
Samll sample volume (1-5 µL)

26. 2. 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

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

28. Non-specific interactions
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

29. 4. 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
Not suitable for absolute quantitative analysis of various oligomers

30. 5. Comparison of NanoDLSay™ with colorimetric assay
Target analyte binding-induced AuNP cluster formation causes SPR band shift of AuNPs to longer wavelength
- Color change Y
Color change
Colorimetric assay
Easy to perform
With or without instrument
Low sensitivity
Does not reveal molecular size information
Not suitable for colored samples (e.g. blood)
Wavelength (nm)
Absorbance (A.U.)
400
800
Before assay
After assay
600
500
700

31. Product & Services Part IV.
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

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

33. 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