NanoDLSay™: Nanoparticle-Enabled Dynamic Light Scattering Assay for Chemical and Biological...
33
NanoDLSay™: Nanoparticle-Enabled Dynamic Light Scattering Assay for Chemical and Biological Detection and Analysis Nano Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954 Email: sales@nanodiscovery July 2012 One New Technology, Discover a New World
NanoDLSay™: Nanoparticle-Enabled Dynamic Light Scattering Assay for Chemical and Biological Detection and Analysis Copyright Nano Discovery Inc. 2012
NanoDLSay: Nanoparticle-Enabled Dynamic Light Scattering Assay
for Chemical and Biological Detection and Analysis Copyright Nano
Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954
Email:
[email protected]@nanodiscoveryinc.com
July 2012 One New Technology, Discover a New World
Slide 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
Slide 3
Part I. General Introduction o The principle of NanoDLSay o How
to conduct NanoDLSay o Applications and examples o Analytical
performance and advantages
Slide 4
What is NanoDLSay: Detect the target analytes by monitoring the
size change of nanoparticles upon binding with the target analyte
AuNP immunoprobe D 120 nm AuNP immunoprobe bound with a small
protein monomer D 130-160 nm AuNP immunoprobe bound with a large
protein complex D > 130-160 nm Y Y Y Y Y Y Y Y Y Y Y Y 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+ Y Y Y
Y Y Y Y General assay format: AuNP clusters formed from binding
with target analytes D >> 100-200 nm Gold nanoparticle
(AuNP)
Slide 5
Two assay formats Y Y Y Y Y Y Y I. Individual particle size
increase Suitable for large analytes such as proteins, complexes
and viruses Suitable for kinetic binding studies II. Nanoparticle
cluster formation Suitable for any analytes, especially for
chemicals and ions Provide best sensitivity Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Slide 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
Slide 7
Why Gold Nanoparticles (AuNPs)? Exceptionally intense light
scattering property 10 5 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 AuNPs 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)
Slide 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 A typical assay condition: 1.Mix 40 L AuNP probe with 2 L
sample 2.Incubate 5-15 min at room temperature 3.Analyze the
particle size to obtain results Read-out: average particle size
(nm) Dose-response curve Target concentration Average particle size
(nm) Unknown sample Standard curve
Slide 9
AuNP Bioconjugate Preparation 1. Direct adsorption method
Citrate AuNPs (100 nm) (Ted Pella Inc.) (1 mL AuNP + 5-10 g
antibody) Blocking reagent: Bovine serum albumin (BSA) (2.5 mg/mL)
1.Centrifuge 2.Re-dispersion ~15 min ~30 min 2. Covalent
conjugation method -NH 2 or -COOH NHS/EDC activation 1.Centrifuge
2.Re-dispersion Functional ligand-coated AuNPs Easy to use but
lower stability; primary choice More complicated but higher
stability
Slide 10
Applications of NanoDLSay Proteins DNAs RNAs Viruses Small
chemicals Toxic metal ions
Slide 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;
130:2780-2782. 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, 11047-11049. Driskell JD, et al. One-step assay for
detecting influenza virus using dynamic light scattering and gold
nanoparticles. Analyst 2011; 136:3083-3090. 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. 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.comwww.nanodiscoveryinc.com
Slide 12
Analytical Performance AnalytesSensitivityDynamic Range
ProteinsHigh pg/mL to low ng/mL range2-3 orders of magnitude DNAs30
fM (5 orders of magnitude more sensitive than SPR and fluorescence
techniques) > 5 orders of magnitude Viruses< 100 TCID 50 /mL
(1-2 orders of magnitude more sensitive than commercial diagnostic
kits) 2-3 orders of magnitude Toxic metal ionsArsenics: 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
molecules7 nM (5 orders of magnitude more sensitive than the
colorimetric method) > 4 orders of magnitude Explosive
chemicals100 pM2-3 orders of magnitude Notes: (1) ng-nanogram;
fg-femtogram; fM-femtomolar; pM-picomolar; nM-nanomolar; ppb-parts
per billion; ppt-parts per trillion; TCID 50 - 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.
Slide 13
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. LabelMethodDetection limit AuNPColorimetric1 10 -8
mol/L Au chipSurface plasmon resonance1 10 -9 mol/L Au/polyaniline
nantube Electrochemical impedance spectroscopy 3 10 -13 mol/L
Quantum dotsAnodic stripping voltammetry5 10 -11 mol/L ZnS and CdSe
quantum dots Fluorescence2 10 -9 mol/L NanoDLSayDynamic light
scattering3 10 -14 mol/L Comparison of NanoDLSay with other methods
for DNA detection Analytical Performance
Slide 14
The ultrahigh sensitivity of NanoDLSay AuNP monomer versus
clusters Size100 nm~300 nm Scattered light intensity ratio1~1000
Number (molar) ratio99.9% (10 pM)0.1% (10 fM) Net scattered light
intensity11 Intensity-averaged particle size * 100 nm + * 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
Slide 15
Advantages of NanoDLSay o Requires a small volume of sample
(1-5 L) 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
Slide 16
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
Slide 17
Introduction: Understand the problems of traditional
immunoassay X B Traditional immunoassay likely fails to detect
proteins in complexes A Traditional immunoassay assumes proteins
exist alone antibody Individual protein monomer Protein complex
Protein aggregates A protein does not stay alone in biological
systems
Slide 18
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
NanoDLSay : Detect target proteins in all forms Unique
capabilities
Slide 19
Dose-response curve Target concentration Average particle size
(nm) Unknown sample Standard curve 1. Protein detection and
concentration analysis Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Single-probe
assay 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 Y Y Y Y Y Y Y
Slide 20
2. Kinetic study of protein-protein interaction Target A Target
B 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 (nm) Incubation time (min) 0
30 Non-binding proteins Alternative:
Slide 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
Slide 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
Slide 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: 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. Snchez-Pomales G, et al. A
lectin-based gold nanoparticle assay for proving glycosylation of
glycoproteins. Biotechnology Bioengineering 2012, published online.
Wang, X.; Ramstrm, 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.
Slide 24
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
Slide 25
1. NanoDLSay versus ELISA Sandwich ELISA NanoDLSay 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 L) 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 L)
Slide 26
2. NanoDLSay versus Surface Plasmon Resonance (SPR) o
Label-free technique o Optical substrate: gold nanoparticle o
Read-out: AuNP size change o Homogeneous solution assay o Low cost
of consumables o Reveal the size information of the target analyte,
distinguish protein complexes and oligomers/complexes from monomers
o Label-free technique o Optical substrate: gold thin film o
Read-out: refractive index change o Heterogeneous chip assay o High
cost of consumables o Does not reveal the size information of the
target analyte, does not tell whether a protein is a monomer,
complex or oligomer NanoDLSay SPR
Slide 27
3. Comparison of NanoDLSay with co-immunoprecipitation (Co-IP)
followed by immunoblotting for protein complex analysis
Slide 28
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
Slide 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: 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
Slide 30
5. Comparison of NanoDLSay with colorimetric assay Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Color change Wavelength
(nm) Absorbance (A.U.) 400 800 Before assay After assay 600 500 700
Colorimetric assay o Easy to perform o With or without instrument o
Low sensitivity o Does not reveal molecular size information o Not
suitable for colored samples (e.g. blood) Target analyte
binding-induced AuNP cluster formation causes SPR band shift of
AuNPs to longer wavelength - Color change
Slide 31
Part IV. NDS1200: A new dynamic light scattering instrument
designed for performing NanoDLSay Product & Services Automatic
measurement of 12 samples Automatic kinetic study of 12 samples
Fast analysis time: 10-20s per sample 40 L 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: 120- 180 samples/hour
The hardware is maintenance-free No special housing environment is
required for the instrument Extremely easy-to-use software
Slide 32
Product & Services NanoDLSay software: A software designed
for convenient, flexible and high throughput analysis
Slide 33
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 Please Contact Us to Request a
Quote: NDS1200 Dynamic light scattering instrument for conducting
NanoDLSay Assay kit including disposable sample cells and other
consumables 3251 Progress Drive Suite A1 Orlando, FL 32826 Phone:
407-770-8954 Email: [email protected] NDS-Kit1000 Order
Information Or visit online: www. nanodiscoveryinc.com Product and
Order Information