1. One new technology, discover a whole new world
NanoDLSay™
Nanoparticle-Enabled Dynamic Light Scattering Assay for Biological and Chemical Detection and Analysis
12565 Research Parkway Suite 300
Orlando, Florida
Copyright Nano Discovery Inc. 2012

2. One new technology, discover a whole new world
NanoDLSay™: a new analytical technique that uses particle size change for signal transduction
Gold nanoparticles (AuNPs) are used as the optical probe in NanoDLSay™

3. A protein complex
(> 20 nm)
Immunoglobulin G
(IgG) (~7-10 nm)
Viruses (~10s nm)
Liu X, Dai Q, Austin L, Coutts J, Knowles G, J, Chen H, H Q. A One-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering. J. Am. Chem. Soc. 2008; 130:
2. Dai Q, Liu X, Coutts J, Austin L, Q. A one-step highly sensitive method for DNA detection using dynamic light scattering. J. Am. Chem. Soc. 2008; 130:
When analytes are bound to the nanoparticles, this will cause various changes to the particle size…
Citrate-protected AuNP ( nm)
AuNP immunoprobe
D » 120 nm
AuNP immune probe 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+
A small chemical
(< a few nm)
A protein monomer (~5-20 nm)

4. NanoDLSay™ Procedure One-step homogenous solution assay
Required sample volume: 1-5 µL
Results obtain in a few minutes

5. APPLICATIONS NanoDLSay™ Proteins DNAs RNAs Viruses Toxic metal ions
Small
chemicals
Toxic
metal ions

6. Publications Protein detection and protein cancer biomarker research:
Liu X, Dai Q, Austin L, Coutts J, Knowles G, Zou J, Chen H, Huo Q. A One-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering. J. Am. Chem. Soc. 2008; 130:
Liu X, Huo, Q. A washing-free and amplification-free one-step homogeneous assay for protein detection using gold nanoparticle probes and dynamic light scattering. J. Immun. Method 2009; 349:
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:
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,
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:
Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:
Huo Q, Colon J, Codero A, Bogdanovic J, Baker CH, Goodison S, Pensky MY. A facile nanoparticle immunoassay for cancer biomarker discovery. J. Nanobiotechnology 2011; 9:20 (open access).
Jaganathan S, Yue P, Paladino DC, 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).
Chun C, Joo J, Kwon D, Kim CS, Cha HJ, Chung MS, Jeon S. 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, 
Wang, X.; Ramström, O.; Yan, M. Dynamic light scattering as an efficient tool to study glyconanoparticle- lectin interactions. Analyst 2011, 136,

7. Publications DNA detection: Virus detection: Small chemical detection:
Dai Q, Liu X, Coutts J, Austin L, Huo Q. A one-step highly sensitive method for DNA detection using dynamic light scattering. J. Am. Chem. Soc. 2008; 130:
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. 
Miao XM, Xiong C, Wei WW, Ling LS, Shuai XT. Dynamic light scattering based sequence-specific recognition of double-stranded DNA with oligonucleotide functionalized gold nanoparticles. Chem. Eur. J. 2011, 17, 
Pylaev TE, Khanadeev VA, Khlebtsov BN, Dykman LA, Bogatyrev VA, Khlebtsov NG. Colorimetric and dynamic light scattering detection of DNA sequences by using positively charged gold nanospheres: a comparative study with gold nanorods. Nanotechnology 2011; 22: (11pp)
Virus detection:
Driskell JD, Jones CA, Tompkins SM, Tripp RA. One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst 2011; 136:
Small chemical detection:
Yang X, Huang J, Wang Q, Wang K, Yang L, Huo X. A one-step sensitive dynamic light scattering method for adenosine detection using split aptamer fragments. Anal Method 2011; 3: 
Dasary SSR, Senapati D, Singh AK, Anjaneyulu Y, Yu H, Ray PC. Highly sensitive and selective dynamic light scattering assay for TNT detection using p-ATP attached gold nanoparticles. ACS Appl. Mater. Interface 2010; 2: 

8. Publications Toxic metal ion detection:
Kalluri JR, Arbneshi T, Khan SA, Nelly A, Candice P, Varisli B, Washington M, McAfee S, Robinson B, Banerjee S, Singh AK, Senapati D, Ray PC. 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:
Beqa L, Singh AK, Khan SA, Senapati D, Arumugam SR, Ray PC. Gold nanoparticle-based simple colorimetric and ultrasensitive dynamic light scattering assay for the selective detection of Pb(II) from paints, plastics, and water samples. ACS Appl. Mater. Interfaces 2011; 3:
Miao X, Ling L, Shua X. Ultrasensitive detection of lead(II) with DNAzyme and gold nanoparticles probes by using a dynamic light scattering technique. Chem. Comm. 2011; 47:
Zhang L, Yao Y, Shan J, Li H. Lead (II) ion detection in surface water with pM sensitivity using aza- crown-ether-modified silver nanoparticles via dynamic light scattering. Nanotechnology 2011; 22: (8pp)
Miao X, Ling L.; Shuai X. Detection of Pb2+ at attomole levels by using dynamic light scattering and unmodified gold nanoparticles. Anal. Biochem. 2012, 421,
Xiong C, Ling L. Label-free, sensitive detection of Hg(II) with gold nanoparticles by using dynamic light scattering technique. Talanta 2012, 89,

9. 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.

10. 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.

11. Advantages NanoDLSay™ Extremely simple Ultra-high sensitivity
Excellent reproducibility
Homogeneous solution assay
Extremely easy to learn and use
Data is easy to collect and interpret
Require small volume of sample (<5 µL)
No special sample preparation is required
Applicable to a wide range of sample matrices
Ultra-low cost of consumables to conduct the assay
Results are obtained in minutes instead of hours or days
Reveals new molecular information that cannot be or has not been detected by any other existing techniques
NanoDLSay™

12. One new technology, discover a whole new world
NanoDLSay™: The most comprehensive and powerful tool available for protein detection and analysis

13. X Limitations of traditional ELISA Comparisons A B
Individual protein monomer
Protein complex
Protein aggregates A B
antibody X
Suitable for detecting individual proteins
Not suitable when protein complexes are present

14. NanoDLSay™
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

15. Particle size change upon antibody addition
NanoDLSay™
A two-step assay for protein complex detection and binding partner analysis
Average particle size increase (nm)
Incubation time (min)
Step 2: Antibody screening
Step 1: Catch the target
Particle size change upon antibody addition c
 ~ 2D
Binding partners
Not binding partners
0 min
60 min

16. Co-Immunoprecipitation
NanoDLSay™: a two-step process
Completed in ~30 min
Co-Immunoprecipitation (Co-IP):
Multiple steps (approximately steps)
Takes hours to days to complete

17. Non-specific interactions
Co-IP and NanoDLSay™:
Which one is more specific?
Centrifuge in Co-IP process increases non- specific interactions
Non-specific proteins are “caught” by the particles due to increased particle concentration towards the end of centrifuge
This problem is not present in NanoDLSay™
NanoDLSay™ is more specific than Co-IP
Centrifuge

18. Label-free detection
NanoDLSay™ for label-free protein oligomer and aggregate detection from real biological samples Y Y
NanoDLSay™
Other techniques
Fluorescence techniques require the labeling of target proteins
Size exclusion chromatography (SEC) and analytical ultracentrifuge (AU) are only suitable for pure protein solution study
SEC may underestimate the aggregation level due to solvent elution; while AU may overestimate the aggregation level due to centrifuge-induced enrichment
Label free
Detects protein oligomers and aggregates directly from real biological samples
Does not change the aggregation status of the sample during the assay
High to ultra-high sensitivity

19. Product & Services
NDS1200: A new dynamic light scattering instrument designed for NanoDLSay™
Automatic measurement 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 capability: samples/hour
The hardware is maintenance-free
No special housing environment is required

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

21. Order Information NDS1200 NDS-Kit100
Dynamic light scattering instrument for conducting NanoDLSay™
NDS-Kit100
Assay kit including disposable sample cells and other consumables
Please Contact Us to Request a Quote:
12565 Research Parkway Suite 300
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