Applications
Biofonctionalization
of
gold nanoparticles
Bio applications of gold nanoparticles:
1. Sensing, e.g. point of care diagnostics and ultrasensitive
biodetection of pathogens.
2. Therapeutic, e.g. targeted drug delivery and photothermal
destruction of malignant cells.
3. Imaging, e.g. nanoparticles as biomolecular probes.
Dr Raphaël Lévy
BBSRC David Phillips Fellow
Phone
+44 151 795 4468
Fax
+44 151 795 4406
Email
[email protected]
Also: non-bio applications of biofunctionalized nanoparticles:
bottom-up nanotechnology, biomimetic approaches to catalysis
and nanoengineering…
Biosciences Building, Crown Street
University of Liverpool, Liverpool L69 7ZB, UK
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« Nanoparticules d'or et résonance de plasmon : des phénomènes physiques aux applications en biologie »
1ere école thématique, 9 au 13 juin 2008, Ile de Porquerolles, Var, France
Optical properties
How?
Physical properties of gold nanoparticles:
Size (nm)
1. Nanoparticle as a template on which to assemble functions
2. Optical properties: plasmon band dependant on the nanoparticle
environment, SERS, resonant scattering, large absorbance crosssection
30
• Scattering
Dark field microscopy, Schultz et al, PNAS 2000
10
3. Optical properties: nanoparticles as a fluorescence quencher
4. Conductivity: nanoparticle as a electronic switch (e.g. Nacardio)
3
• Absorption
5. Electron dense: contrast agent in Electron Microscopy
• Interactions
Dispersed
Agregates
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Also: some very small gold/silver nanoparticles (below 2nm) do
exhibit some fluorescence. Not yet any convincing
bioapplications.
Photothermal microscopy, Boyer et al, Science 2002
Some examples
of applications
Some examples
of applications
Immunoelectron
microscopic
localization of cellular
proteins and
carbohydrates.
(immunoreactivity
is present in the
cytosol and
nucleoplasm of
epithelia of distal
convoluted tubules,
chick kidney (see also
Roth et al. 1981b) (A);
Nature – 15 August 1996 –
p607
Keywords: “gold” and
“immuno”
800
Source: ISI Web of
Knowledge
600
200
Science – 22 August 1997 –
p1078
0
1960
1970
1980
1990
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Gold nanoparticles and their
bioconjugates already have a
long history and have already
made a big impact on
biological research.
400
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Number of publications
1000
DNA to assemble nanoparticles,
Nanoparticles to detect DNA
2000
Year
Some examples
of applications
Some examples
of applications
Nanoparticles to detect DNA
Nanoparticles to detect protease activity in vivo
Seulki Lee et al., Angew. Chem. Int. Ed. 2008, 47, 2804
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A simple solution test based on colour change (plasmon
coupling) allows detection of single bp mismatch
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Science – 22 August 1997 – p1078
Distance-dependent fluorescence quenching allows real-time
in vivo detection of a protease (cancer marker)
Some examples
of applications
The choice of the nanoparticle coating is
critical for the development of any
nanoparticle-based applications.
Single molecule imaging in living cells
The coating has to fulfil several requirements:
1. Providing colloidal stability
In the absence of a stabilizing layer, gold nanoparticles, e.g. citrate synthesis, are stabilized in pure
water by electrostatic repulsion. At high ionic strenght, i.e. in conditions relevant to bioassays, the
electrostatic repulsion is screened and the nanoparticles aggregate due to Van-der-Waals
interactions.
2. Limiting unspecific interactions
In the absence of a stabilizing layer, biomolecules such as DNA and Proteins do interact with gold
non-specifically (VdW, electrostatic). Applications with complex biological systems require to
reduce such interactions to the minimum.
Photothermal microscopy
allows tracking of single
molecule/particle in living
cells over long timecourse
(no photobleaching)
3. Chemical stability
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Thiol chemistry is the easiest and mostly used method to attach molecules to gold nanoparticles.
However, thiols attached to the surface can be replaced for thiols in solution through a reaction
called “ligand exchange”
4. Providing one or several molecular functions (targeting, sensing, etc)
Monolayer Protected Clusters (MPCs)
S
S
S
HAuCl4 . 3H2O
H 2O
TOABr
Au
Au
S
S
S
S
S
S
Aqueous solution of
sodium citrate
Aqueous solution
of HAuCl4
S
S
S
Dodecanethiol/NaBH4
Preparation of
gold hydrosols
S
S
S
S
Toluene
+
J. Turkevich et al., Discuss. Faraday. Soc. 1951, 11, 55.
200 nm
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M. Brust, M. Walker, D. Bethell, D. J. Schiffrin, R. Whyman,
J. Chem. Soc.,Chem. Commun. 1994, 801.
Such gold hydrosols have excellent colloidal stability… as long as
you keep them in pure water.
Functionalization
“New”
Approaches
Classical approach
Immuno-electron microscopists have prepared and
used gold colloids functionalized with antibodies for
the past 5 decades.
BIOTIN
AVIDIN
Protocols for the adsorption of Protein A (which
binds antibodies) can be found on the web or in
methods textbooks, e.g.,
ENZYME
Au
Bioconjugates Techniques, Greg T Hermanson
ANTIBODY
Protein A coated gold nanoparticles and Antibodies coated gold nanoparticles can be
bought from several companies. BBI, British BioCells has a large share of the market
on the sale of colloidal golds and its conjugates.
DNA
http://www.bbigold.com/products/goldconjugates.asp?navid=2
Aim: convenient, robust and versatile synthesis providing multifunctional
nanoparticle with a control over valency and orientation of functional groups
NH2
Oligonucleotides
N
Thiol-terminated oligonucleotides
Mirkin, Niemeyer, Alivisatos
N
O
S
OP O
O
O
H
O
Gold nanoparticles,
e.g. citrate-stabilised
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“New”
Approaches
BSA
H
H
O
H
P
NH2
H N
O-
N
N
N
O
Silica shell
Liz-Marzan, Giersig, Mulvaney
O
H
O
DNA
H
H
O
H
P
O
H
N
O-
N
O
NH
N
NH2
O
H
Thiol-terminated peg and
alcane-peg
Brust
H
OH
H
H
Citrate-stabilised
gold nanoparticles
Hundreds of papers describing applications: bioarrays,
solution assays, nanoengineering
Some limitations: highly charged, balance between DNA
accessibility and packing density, unspecific assembly
and unspecific interactions, thick layer
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Cysteine-terminated
peptides and peptide
conjugates
Levy, Brust, Fernig
H
Oligonucleotides
Oligonucleotides
First demonstration of the
preparation of nanoparticles
with a precise number of
functional molecules.
Cryo TEM shows some nonspecific aggregation with
ssDNA and dsDNA capped
gold nanoparticles.
Some constraints: needs to
be a very large DNA; gel
electrophoresis is not a
preparative method.
Note large interparticle
distance so no (or very small)
colour change.
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Oligonucleotides
Gold@silica
Beautiful synthesis
500 citations, but not many
bio-applications.
Here the nanoparticle is used
essentially as a vehicle to
modify a cellular function.
Potentially interesting for
SERS probes with the dye
incorporated between the
silica and the gold.
Note multiple thiols to
evaluate the effect of ligand
exchange.
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Silica@
Silver@Silica
Thioalkyl-PEG
OH
O
O
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
OH
Peptides SAM
OH
Particles initially prepared in solvent - transferred in H2O.
Advantageous for synthesis of small nanoparticles. Endgroup can be modified to facilitate further functionalization.
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Peptides SAM
HS
SH
OH
Ag/Au nanoparticule
as a protein core analog
In globular proteins the “hydrophobic core”
Formation of peptide monolayer protected particles
Suitable peptide
-
holds the surface residues in the correct position
-
-
Peptides as capping ligands ?
-
-
Citrate stabilised
gold nanoparticles
Peptide capped
gold nanoparticles
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Rational Design
CALNN
COO-
O
CH
CH2
hydrophilic
surface
NN
C
O
C CH2
NH2
C
asparagine (N)
NH2
N
O
CH
O C
CH2
CH
leucine (L)
CH3
N
C
CH3
Size exclusion
chromatography
CH3
CH
hydrophobic
core
AL
Ionic strength
asparagine (N)
N
O
CH
alanine (A)
N
(A) PBS pH 7 – (B) PBS 1M NaCl pH 7
O C
anchor
C
(C) PBS 1.25M NaCl pH 7 - (D) PBS 1.5M NaCl pH 7
CH
+
H3 N
CH2
Affinity
chromatography
cysteine (C)
S
pH
Stable in a large range of pH and
ionic strength. Compatible with
molecular biology tools.
CALNN forms a densely packed SAM on Au/Ag nanoparticles
The sequence
space…
Combinatorial
design
5 amino acids Æ 3 200 000 possible sequences
Length
Exploring the peptide sequence space
C O
O
C CH2 CH
NH2
N
O C
CH3
CH CH2 CH
CH3
N
C
O C
Test the design criteria
C
O
O C
O
Improve stability
CH
CH2
+
H3N
S
CA
CAL
CALN
CALNN
Anchor
Core
Core
Terminal
Substitution in
position 1, and
reverse sequence of
CALNN
Hydrophobic
residues substituted
in positions 2 and 3
Hydrophilic
residues substituted
in positions 2 and 3
Substitution in
positions 4 and 5
KALNN
AALNN
CALNN
CCALNN
NNLAC b
NNLAC a, b
CILNN
CLLNN
CVLNN
CFLNN
CAANN
CAINN
CAVNN
CAFNN
CLANN
CKLNN
CDLNN
CTLNN
CNLNN
CAKNN
CADNN
CATNN
CANNN
CDDNN
CKKNN
CTTNN
CTSNN
CALLS
CALLD
CALLK
CALLR
CALNS
CALND
CALNK
CALNR
CALSS
CALSD
CALSK
CALSR
CALKS
CALKD
CALKK
CALLS
CALSS
CALKK
CALNN
b
b
b
b
b
b
New Design
CTTTT
CHRIS b
CVVIT
CCVVVT
CCVVVK b
CAALPDGLAAC a, b
CVVITPDGTIVVC
b
NNLACALNN
NNLACCALNN
b
b
b
b
a, b
a, b
a, b
a, b
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CH
CH2
+
H3N
S
CH3
CH CH2 CH
CH3
N
CH3 CH
N
CH3 CH
N
O C
COOO
CH CH2 C
NH2
N
C O
O
C CH2 CH
NH2
N
COOO
CH CH2 C
NH2
N
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gold nanoparticle
Combinatorial
design
peptides
buffer
NaCl
Lévy et al
JACS 2004, 126, 10076
• Rational design principles are confirmed
• Rules for peptide sequences inducing nanoparticles aggregation
How to introduce
function?
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NaCl
• Roles of thiol, hydrophobic interactions, charge and hydrogen
bonding are defined
NaCl
Matrix peptide (100 - a) % CALNN
Functional peptide
Biotin and
Streptag II
a % CALNNXXXXXX
• Biotin
CALNNGKbiotinG
• Strep-Tag II
CALNNWSHPQFEK
• DNA
CALNN-DNA
• nano1
CALNNGVEAFEKKVA
• NTA, trisNTA
CALNN-NTA
• Histag-biotin
CALNNGH6GKbiotinG
• Loop formation
CALNNGKGAIQGRGKbiotinTAK
• CaM KII substrate
CALNNAALRRASLG
• PKA substrate
CALNNAAKKLNRTLSVA
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• Oligosaccharides, enzymes, etc.
• In preparation:
CALNNGH6GBenzylguanine
Multifunctional
particles
Controlling
valency [1]
• biotin
CALNNGKbiotinG
• DNA
CALNN-DNA
Avidine
Avidine
Protein A
0
Mismatch
3
2
Matrix peptide (100 - a) % CALNN
Functional peptide
a % CALNNGHHHHHH -LABEL
Wang et al, Bioconj
Chem 2005, 16, 497
Controlling
valency [3]
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DNA microarray
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Controlling
valency [2]
a % CALNN-DNA
1
Wang et al, Bioconj
Chem 2005, 16, 497
Protein microarray
Matrix peptide (100 - a) % CALNN
Functional peptide
Matrix peptide (100 - a) % CALNN
Functional peptide
a % CALNNGH6GKbiotinG
Affinity chromatography
Transition from Zero to
One label per nanoparticle
Lévy et al (2006)
Chembiochem 7, 4, 592
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Percentage of functional peptide %
Nanoparticles as kinase
substrates; screening for
kinase inhibitors
Kinase
assay
Peptide-capped gold enter into
cells through endocytosis
Cy3
anti-Flag
1
2
3
4
5
10nm Gold nanoparticles
95% CALNN, 5% FLAG-tag
PKA
Immuno
Fluorescence
DIC
EM
CaMKII
1.
Control (no kinase)
2.
Control (no inhibitor)
3.
Reaction with SB203580
4.
Reaction with KN62
5.
Reaction with H89
But:
1. They are stuck in
endosomes
2. They lose their
functional peptide…
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Zhenxin Wang
Enzyme
assay [2]
ZffmK
CALNNThrFluor
Thrombin cleavage
+
CALNN
QUENCHED
Fluorescence Intensity
3500
3000
2500
2000
1500
1000
500
0
0
10
20
30
40
50
60
Time (mins)
Violaine See, Paul Free,
2008, in preparation.
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Cathepsins cleave the peptide layer inside endososomes in living cells
Monitoring
nanoparticle uptake
Photothermal
microscope for longterm imaging of
nanoparticles in live
cells
Transmission image
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Photothermal image
10 nm CALNN-capped nanoparticles, 3h incubation time
Structural
nanotechnology
Proximity probe
• Peptide SAMs provide an enormous easily
accessible chemical space
•Mobility of thiols – potential for self-organization
Need for new
structural probes…
Self-organization in
peptide SAM ?
• Nanozymes (Scrimin, Pasquato)
• « Templatable » surfaces (Rotello)
• Self-organization –first vizualization on gold
nanoparticles from Stellacci’s group
Proximity probe:
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Range of molecular ruler: MD simulations
Proximity probe
Without BS3
With BS3
Duchesne et al, 2008, ChemBioChem, in press
Biosciences, Liverpool
• Yann Cesbron, Chris Shaw, Umbreen Shaheen, Paul Free
• Violaine See
• Dave Fernig, Laurence Duchesne
Chemistry, Liverpool
• Mathias Brust, Zhenxin Wang
Physics, Leeds
• Sarah Harris
Physics, Bordeaux
• Brahim Lounis
• Jean-Pierre Aime
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Intermolecular cross-linking occurs at very low surface coverage
Î Experimental results are not compatible with a random
localization of the functional peptides but rather indicate that
the functional peptides self-organize into a dense patch or
supramolecular domain
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