gold nanoparticles - optimization of conjugates

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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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