NanoDLSay™Nanoparticle-Enabled Dynamic Light Scattering Assay

for Biological and Chemical Detection and Analysis

12565 Research Parkway Suite 300Orlando, Florida

Email: huo@nanodiscoveryinc.com

One new technology, discover a whole new

world

www.nanodiscoveryinc.com

Copyright Nano Discovery Inc. 2012

NanoDLSay™: a new analytical

technique that uses particle size

change for signal transduction

Gold nanoparticles (AuNPs) are used as the optical probe in NanoDLSay™

One new technology, discover a whole new

world

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; 130:2780-2782.2. 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: 8138-8139.

Citrate-protected AuNP (10-100 nm)

A protein monomer (~5-20 nm)

A small chemical(< a few nm)

AuNP immunoprobe

D » 120 nm

AuNP immune probe bound with a small protein monomer

D » 130-160 nm

AuNP immunoprobe bound with a large protein

complex

D > 130-160 nm

Y

YY

YY

Y

Y

Y

Y

Y

YY

Y

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+

2+

2+

2+

Y

Y

Y

YY

Y

Y

A protein complex (> 20 nm)

Immunoglobulin G (IgG) (~7-10 nm)

Viruses (~10s nm)

When analytes are bound to the nanoparticles, this will cause various changes to the particle size…

NanoDLSay™ Procedure

One-step homogenous solution assay Required sample volume: 1-5 µL Results obtain in a few minutes

NanoDLSay™

Proteins

DNAsRNAsViruses

Small chemicals

Toxic metal ions

APPLICATIONS

PublicationsProtein 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; 130:2780-2782.

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: 38-44.

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.

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.

Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:259-265.

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, 11047-11049.

Wang, X.; Ramström, O.; Yan, M. Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions. Analyst 2011, 136, 4174-4178.

Publications

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; 136:3083-3090.

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; 3:59-61.

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; 2:3455-3460.

DNA 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: 8138-8139.

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, 11230-11236.

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; 22:285501 (11pp)

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; 48:9668-9671.

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; 3:668-673.

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; 47:4192-4194.

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; 22:275504 (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, 582-586.

Xiong C, Ling L. Label-free, sensitive detection of Hg(II) with gold nanoparticles by using dynamic light scattering technique. Talanta 2012, 89, 317-321.

Analytical Performance

Analytes Sensitivity Dynamic RangeProteins 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)

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

Explosive chemicals

100 pM 2-3 orders of magnitude

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.

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.

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

Comparison of NanoDLSay™ with other methods for DNA detection

Analytical Performance

Advantages

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™

NanoDLSay™: The most comprehensive

and powerful tool available for protein

detection and analysis

One new technology, discover a whole new

world

X

Individual protein monomer

Protein complex Protein aggregates

A B

Limitations of traditional ELISA

Comparisons

Suitable for detecting individual proteins

Not suitable when protein complexes are present

antibody

Avera

ge p

art

icle

siz

e incr

ease

(nm

)

Incubation time (min)

= 2D of analyte

0 min 30 min

1

2

3

NanoDLSay™

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

NanoDLSay™

A two-step assay for protein complex detection and binding partner analysis

Avera

ge p

art

icle

siz

e incr

ease

(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

Co-Immunoprecipitation NanoDLSay™: a two-step

process Completed in ~30 min

Co-Immunoprecipitation (Co-IP): Multiple steps (approximately 15-20 steps) Takes hours to days to complete

Centr

ifuge

Non-specific interactions

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

Co-IP and NanoDLSay™:Which one is more specific?

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

NanoDLSay™ for label-free protein oligomer and aggregate detection from real biological samples

Label-free detection

NanoDLSay™

YY

Y

YY

Y

Y

Y

Y

Y

YY

Y

Y Y

Y

YY

Y

Y

Y

Y

Y

YY

Y

Y

Y

Other techniques

NDS1200: A new dynamic light scattering instrument designed for NanoDLSay™

Product & Services

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: 120-180 samples/hour

The hardware is maintenance-free No special housing environment is

required

Product & Services

NanoDLSay™ software: A software designed for convenient, flexible and high throughput analysis

Notes

Patent application pending on NanoDLSay™ technology and NDS1200 system: PCT/US09/030087 and PCT/US11/21002 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

12565 Research Parkway Suite 300Orlando, FL 32826Phone: 407-770-8954Email: sales@nanodiscoveryinc.com

NDS-Kit100

Order Information

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