gold nanoparticles - optimization of conjugates
DESCRIPTION
In this webinar the CEO and CSO of Innova Biosciences, Dr Nick Gee, provides an in-depth overview of the properties of gold nanoparticles and approaches for creating conjugates with proteins and small molecules. The importance of shape, size and surface chemistry in different applications is also discussed.TRANSCRIPT
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Quality – Consistency – Expertise
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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
YouTube: www.youtube.com/InnovaBiosciences
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