gold nanoparticles - optimization of conjugates

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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




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 nanoparticles display shape- and size-dependent properties.

Gold nanoparticles (10-200nm range)

Light Surface electrons

Gold Gold

--

---

--

++ ++ ++

Plasmonresonance




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 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




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




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

Thiol group

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-thiolMono-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 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




Naked gold conjugates - pH optimisation/salt stability

x

x

x

x




Support grid

Surface layerHole in grid

40nm gold particle

Covalent approach to conjugation with InnovaCoat GOLD

1.5-2 nm thick

TEM analysis




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




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




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.




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

Sig

na

l

InnovaCoat covalent conjugates

passive




Oriented coupling of whole antibodies:

hydrazide

HydrazideInnovaCoatderivative

periodatetreatedantibody




Oriented coupling of antibody fragments

For coupling Fab’ or engineered antibody fragments expressed with a cys terminus.

maleimide +Fab’

No protein:metal contact




Lateral flow data with InnovaCoat GOLD 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

--

---

--

++ ++ ++

Plasmonresonance

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 plasmon shift 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

InnovaCoat streptavidin

+ InnovaCoat biotin

+ biotin

+ biotin - biotin




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




FRET quenching/Metal enhanced fluorescence

Raman Quenching (0-5nm)

Enhancement (10-30nm)Silver (gold)

Fluorescent molecule




Photothermal therapy

Infrared laser

tumour

energy

Hollow sphereSilica 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 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)




Contact

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