Gold nanoparticles - optimization of conjugates
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Transcript of Gold nanoparticles - optimization of conjugates
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Quality – Consistency – Expertise
© Innova Biosciences Ltd. All rights reserved
Gold nanoparticles:optimization of conjugates and the importance of size, shape and surface properties in different applications
Speaker: Dr Nick Gee
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Dr Nick Gee, CEO/CSO
• the size and shape of gold nanoparticles
• how to make conjugates using passive binding and covalent conjugation techniques
• and shape/size requirements
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Bulk gold 40nm nanoparticles
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Gold nanoparticles display shape- and size-dependent properties.
Gold nanoparticles (10-200nm range)
Light Surface electrons
Gold Gold
--
---
--
++ ++ ++
Plasmonresonance
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50nm
TEM data for commercially available “40nm” nanoparticles
The best technique for studying the shape of individual particles
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Ratio of A650:A530 is a useful “aggregation parameter”
Absorbance scanning of naked gold nanoparticles
- + NaCl
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Particles with high sphericity will have PW75 values of 45nm or less.
Monodisperse spherical particles have an A650:A530 ratio of < 0.1.
Peak position is a crude measure of particle diameter. Peak absorbance for 40nm gold in water is at ~526 nm.
PW75
Information on sphericity can obtained from scans
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400 450 500 550 600 650 7000
25
50
75
100
Wavelength (nm)
Ab
so
rban
ce %
PW75 Sample PW75 value (nm) 650/530 ratio
1 63 0.16
2 56 0.16
3 45.5 0.13
4 46.5 0.12
5 50 0.24
Comparison of nanoparticles by absorbance scanning
Quality parameters
Performance in different applications may not be the same
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Methods of attaching molecules to gold nanoparticles
Two main types of material (naked, coated), three approaches:
(i) Self-assembly of small thiolated molecules on naked gold(ii) Passive adsorption of proteins on naked gold(iii) Covalent attachment of (bio)molecules on coated gold
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Attachment of small molecules to gold surfaces
Thiol group
Thiolated (-SH) molecule (or disulfide)
Au-S dative bond
Organic surface
metal
X X X X X Functional group
Self assembly technique
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Self assembly with nanoparticles
--
---
--
-
--
Citrate ions
dissociation
+
Naked gold
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Reducing the rate of ligand dissociation
metal
X X X X X
metal
X X X
Di-thiolMono-thiol
X = drug or functional group (e.g. COOH)
Stronger interaction
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• High risk of aggregation if the conditions are wrong.
• Binding is pH and salt dependent (pH = isoelectric point + 0.5).
• Need to carry out multiple trial conjugations.
• Conjugate stability is checked in a stress test with NaCl.
Attachment of antibodies to naked gold
• Binding mechanism is not completely understood.
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40nm diameter by TEMPW75 value = 43nmA650/530 ratio < 0.1
400 450 500 550 600 650 7000
10
20
30
40
50
60
70
80
90
100
Ab
sorb
ance
%
Studies with naked gold (Innova GOLD)
43 nm
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Naked gold conjugates - pH optimisation/salt stability
x
x
x
x
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Support grid
Surface layerHole in grid
40nm gold particle
Covalent approach to conjugation with InnovaCoat GOLD
1.5-2 nm thick
TEM analysis
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Properties of InnovaCoat GOLD
Protective coat shields the metal from the external environment. Resistant to high and low pH, and high salt concentration. Survives 2.5M NaOH at 70oC for >90 min. Cannot be used for passive binding.
Naked Coated
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Functionality of InnovaCoat GOLD
Irreversible attachment of analytes and antibodies
Amine, H2N Carboxyl
Maleimide
Hydrazide Biotin
Streptavidin
Amines/CDI(lysine)
Thiols(cysteine)
Streptavidinylatedmolecules
Aldehydes(periodate treatedantibodies)
Biotinylatedmolecules
NHS esters,COOH/CDI
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Special one-step conjugation kits for antibodies
Freeze dried InnovaCoat gold plus all required chemicals1-2 min hands-on time, conjugate ready in 20 min.
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Passive binding versus covalent attachment
Which is the better approach? It depends……
Whole antibodies (easier than passive).Small molecules (irreversible link).Antibody fragments (Fab).Entities that require a cleavable link.Multiple chemistries.Generally requires less antibody.
Naked InnovaCoat
Whole antibodies (‘traditional’ passive approach).Small thiolated ligands (exchangeable).
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CRP conjugates – passive versus covalent in lateral flow
CRP1
CRP1
CRP2
CRP2
CRP3
CRP3
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
Sig
na
l
InnovaCoat covalent conjugates
passive
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Oriented coupling of whole antibodies:
hydrazide
HydrazideInnovaCoatderivative
periodatetreatedantibody
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Oriented coupling of antibody fragments
For coupling Fab’ or engineered antibody fragments expressed with a cys terminus.
maleimide +Fab’
No protein:metal contact
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Lateral flow data with InnovaCoat GOLD coupled to Fab’
(coupling to COOH surface coat)
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Overview of tools and conjugation methods
• Gold nanoparticles come in many different shapes and sizes.
• Can attach molecules by passive adsorption, self assembly, or irreversible anchoring.
• Simple one-step covalent methods available for antibodies.
• Range of covalent chemistries available for other analytes.
• Best approach determined by nature of substance to be conjugated and the intended application.
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Properties of gold nanoparticles
Gold
--
---
--
++ ++ ++
Plasmonresonance
Absorption (colour)
Electric field created
Altered by the environment (type of solvent, other molecules or particles in close proximity)
Light
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Snapshot of applications of gold nanoparticles
Lateral flow tests (major use)
SERS
Surface plasmon shift assays
Metal-enhanced fluorescence
Detection Therapeutics
Microscopy
Infra-red phototherapy
Radio-frequency ablation
Drug delivery/targeting
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Plasmon shift assays
Red>blue colour change
Regular dispersed spheres best
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Bio-sensing assay format
InnovaCoat streptavidin
+ InnovaCoat biotin
+ biotin
+ biotin - biotin
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SERS - surface enhanced Raman spectroscopy
Raman spectroscopy provides information about vibrations
Raman shift cm-1
500 20001000 1500
Intensity
Intense field between gold particles
Raman dye
Nanoparticles, or nanoscale surface defects
Rough surface
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FRET quenching/Metal enhanced fluorescence
Raman Quenching (0-5nm)
Enhancement (10-30nm)Silver (gold)
Fluorescent molecule
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Photothermal therapy
Infrared laser
tumour
energy
Hollow sphereSilica shell
rod
Infrared light has better penetration of tissues
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Non-invasive radiofrequency ablation
RF
Nanoparticles injected Tumour
Current RF therapy is invasive and uses electrodes inserted into tissue
Radiofrequencies are safe and easily penetrate tissues
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Applications – size and shape requirements
5 10 20 40 60 80 Shape SurfaceLateral flow Sphere
Western blots SphereDot blots Sphere
Dark field microscopy SphereElectron microscopy Sphere
Metal enhanced fluorescence Sphere/flat surface Rough/spikySERS Not spherical Rough/spiky
FRET quench SphereInfrared phototermal therapy Rod/hollow sphere
Radiofrequency ablation SphereDrug delivery Rod/sphere
Size (nm)
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Contact
If you would like any more information, please contact us at [email protected]
Please keep an eye out for our future webinars and other exciting news on our website and social media channels:
www.innovabiosciences.com/innova/webinars.html
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