Gold nanoparticles - optimization of conjugates

© Innova Biosciences Ltd. All rights reserved
Quality –Consistency –Expertise

Gold nanoparticles:
optimization of conjugates and the importance of size, shape and surface properties in different applications
Speaker: Dr Nick Gee

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

Bulk gold
40nm nanoparticles

Gold nanoparticlesdisplay shape-and size-dependent
properties.
Gold nanoparticles(10-200nm range)
Light
Surface electrons
Gold
Gold
-
-
-
-
-
-
-
+
+
+
+
+
+
Plasmon
resonance

50nm
TEM data for commercially available “40nm” nanoparticles
The best technique for studying the shape of individual particles

Ratio of A650:A530 is a useful “aggregation parameter”
Absorbance scanning of naked gold nanoparticles
-
+
NaCl

Particles with high sphericitywill
have PW75 values of 45nm or less.
Monodispersespherical particles
have an A650:A530 ratio of < 0.1.
Peak position is a crudemeasure
of particle diameter. Peak absorbance
for 40nm gold in water is at ~526 nm.
PW75
Information on sphericitycan obtained from scans

400 450 500 550 600 650 700
0
25
50
75
100
Wavelength (nm)
Absorbance %
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

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

Attachment of small molecules to gold surfaces
Thiolgroup
Thiolated (-SH) molecule (or disulfide)
Au-S dative bond
Organic surface
metal
X
X
X
X
X
Functional group
Self assembly technique

Self assembly with nanoparticles
-
-
-
-
-
-
-
-
-
-
Citrate ions
dissociation
+
Naked gold

Reducing the rate of ligand dissociation
metal
X
X
X
X
X
metal
X
X
X
Di-thiol
Mono-thiol
X = drug or functional group (e.g. COOH)
Stronger interaction

•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.

40nm diameter by TEM
PW75 value = 43nm
A650/530 ratio < 0.1
400 450 500 550 600 650 700
0
10
20
30
40
50
60
70
80
90
100
Absorbance %
Studies with naked gold (Innova GOLD)
43 nm

Naked gold conjugates -pH optimisation/salt stability
x
x
x
x

Support grid
Surface layer
Hole in grid
40nm gold particle
Covalent approach to conjugation with InnovaCoatGOLD
1.5-2 nm thick
TEM analysis

Properties of InnovaCoatGOLD
Protective coat shields the metal from the external environment. Resistant
to high and low pH, and high salt concentration. Survives 2.5M NaOHat 70oC
for >90 min. Cannotbe used for passive binding.
Naked
Coated

Functionality of InnovaCoatGOLD
Irreversible attachment of analytesand antibodies
Amine, H2N
Carboxyl
Maleimide
Hydrazide
Biotin
Streptavidin
Amines/CDI
(lysine)
Thiols
(cysteine)
Streptavidinylated
molecules
Aldehydes
(periodate treated
antibodies)
Biotinylated
molecules
NHS esters,
COOH/CDI

Special one-step conjugation kits for antibodies
Freeze driedInnovaCoatgold plus all required chemicals
1-2 min hands-on time, conjugate ready in 20 min.

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).

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
Signal
InnovaCoat covalent conjugates
passive

Oriented coupling of whole antibodies:
hydrazide
Hydrazide
InnovaCoat
derivative
periodate
treated
antibody

Oriented coupling of antibody fragments
For coupling Fab’ or engineered antibody fragments
expressed with a cys terminus.
maleimide
+
Fab’
No protein:metalcontact

Lateral flow data with InnovaCoatGOLD coupled to Fab’
(coupling to COOH surface coat)

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.

Properties of gold nanoparticles
Gold
-
-
-
-
-
-
-
+
+
+
+
+
+
Plasmon
resonance
Absorption (colour)
Electric field created
Altered by the environment
(type of solvent, other molecules
or particles in close proximity)
Light

Snapshot of applications of gold nanoparticles
Lateral flow tests (major use)
SERS
Surface plasmonshift assays
Metal-enhanced fluorescence
Detection
Therapeutics
Microscopy
Infra-red phototherapy
Radio-frequency ablation
Drug delivery/targeting

Plasmon shift assays
Red>blue colour change
Regular dispersed
spheres best

Bio-sensing assay format
InnovaCoatstreptavidin
+ InnovaCoatbiotin
+ biotin
+ biotin
-biotin

SERS -surface enhanced Raman spectroscopy
Raman spectroscopy provides information about vibrations
Raman shift cm-1
500
2000
1000
1500
Intensity
Intense field between gold particles
Raman dye
Nanoparticles, or nanoscale surface defects
Rough
surface

FRET quenching/Metal enhanced fluorescence
Raman
Quenching (0-5nm)
Enhancement (10-30nm)
Silver (gold)
Fluorescent molecule

Photothermaltherapy
Infrared laser
tumour
energy
Hollow sphere
Silica shell
rod
Infrared light has better penetration of tissues

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

Applications –size and shape requirements
5
10
20
40
60
80
Shape
Surface
Lateral flow
Sphere
Western blots
Sphere
Dot blots
Sphere
Dark field microscopy
Sphere
Electron microscopy
Sphere
Metal enhanced fluorescence
Sphere/flat surface
Rough/spiky
SERS
Not spherical
Rough/spiky
FRET quench
Sphere
Infrared phototermal therapy
Rod/hollow sphere
Radiofrequency ablation
Sphere
Drug delivery
Rod/sphere
Size (nm)

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
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

Innova Biosciences Ltd. Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova BiosciencesDyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

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