3 Graphene and metal NPs
graphene
metal-based nanostructures
– nanowires, gold nanoparticles (nanorods, nanocages, nanoshells)
magnetic nanoparticles
History
1859 - the first example of chemical exfoliation of graphite
B.C. Brodie treated flakes of graphite with potassium chlorate (KClO3) and fuming
nitric acid (HNO3) at 60 oC for 3–4 days
product washed by water to remove acid
the entire procedure repeated several times until no further change was observed
Brodie managed to isolate material that was “extremely thin and
perfectly transparent”
– resulting material consisted of carbon, oxygen and hydrogen
– which explained the increased total mass from the starting flakes of graphite
“graphic acid” (Brodie’s term) or “ graphitic acid”
– since it was dispersible in neutral and basic media, but not in acids
now typically named “graphite oxide”
– preserves the layered structure of graphite, layers are heavily oxidized
– can be relatively easily separated in water or other polar solvents by mild
sonication or stirring, resulting in a solution of
graphene oxide (GO)
– majority of the flakes are either mono- or few-layer stacks
– can be reduced - hydrazine, hydroxylamine, sodium borohydride, sodium hydride, …
– resulting in “chemically converted”
graphene
single sp2 bonded carbon sheet, C atoms in hexagonal array
Nobel Prize in Physics 2010: Andre Geim
and Konstantin Novoselov “for groundbreaking
experiments regarding the two-dimensional material graphene”
Graphene
Hummer’s method
synthesis of graphite oxide can be accomplished by treating
graphite with a mixture of concentrated sulfuric acid, sodium nitrate
(NaNO3) and potassium permanganate (KMnO4)
– much faster (few hours for completion)
– no explosive gases evolve (… but toxic, yes – caution!)
after hydrazine reduction - TEM image
Graphene oxide
TEM images of graphene (A) and graphene oxide (B); scale bar 2 nm
GO is not conductive
Reduction of GO
not exactly graphene, but rather
reduced GO, rGO
Dispersion of GO
1 h (top) and 3 weeks (bottom) after sonication
Graphene
sp2 hexagonal sheet
orientation
arm-chair / zig-zag
scale bar 0.2 nm
Graphene
STM image, 4x4 nm
hexagonal graphene
unit cell is depicted
AFM image of graphene
from the Hunter’s method
AFM of graphene
left, topography, right, height profile in the indicated line
– observed roughness depends on the supporting material, on mica it could
be around 0.07 nm for a monolayer
Chemically modified graphene
example of modification with the help of diazonium salts in the
presence of ionic liquid
for covalent binding of biomolecules (enzymes), the carboxy
group is particularly useful
Direct oxidation of graphene
should be very careful …
from nanotechweb.org
Graphene in electronics
graphene transistor
Graphene as a transparent electrode
Murali, R. (Ed.). Graphene Nanoelectronics, From Materials to Circuits.
Springer, Heidelber, 2012.
enhanced electron transfer
very simple
mix all together …
more advanced
communication between enzyme active center and
the electrode
Graphene for biosensing
Metal-based
nanomaterials
Type of metal NPs
functionalization:
– anions, polymers,
proteins, …
Synthesis of metallic nanoparticles
the preferred diffusion controlled process can be
achieved by low concentration of solute or polymeric
monolayer adhered onto the growth surface
precursor: elemental metals, inorganic salts and metal
complexes
– anode from Pd / Ni / Co, PdCl2, H2PtCl6, K2PtCl4, HAuCl4,
AgNO3, RhCl3
reduction reagent
– citrate / citric acid, H2O2, hydroxylamine.HCl, H2, CO, P (in ether),
methanol, formaldehyde, NaBH4, NaOH, NH4-, Na2CO3
polymeric stabilizer
– polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene
imine (PEI), polyphosphate, polyacrylate, tetraalkylammonium
halogenides
Gold nanoparticles (AuNP)
preparation:
– chlorauric acid dissolves into water to make 20 ml very dilute
solution of 2.5x10-4M
– then 1 ml 0.5% sodium citrate is added into the boiling solution
– the mixture is kept at 100oC till color changes, while maintaining
the overall volume of the solution by adding water
… Turkevich, J. Discuss. Faraday Soc., 1951
1 nm 2 nm
gold nanoparticle:
about 300 Au atoms
C60 – Buckminsterfullerene
Effect of concentration
Size (nm) Time (min)
Colloid gold
stable suspension of disperged metal particles, size from around 5 to
some 150 nm
– in solution looks intensively coloured (red, corresponding to the size) after
eventual coagulation turns dark (blue)
conjugation with proteins – variable, according to:
– what type of interaction will be responsible for binding with the NP (commonly
negatively charged)
– hydrophobic interaction can be involved, too
– for Au NP (or other noble metal NPs), the strong binding with thiols is
convenient (SAM, self assembled monolayer is formed)
the adsorped proteins generally stabilise the colloid gold and prevent
coagulation
– binding of proteins should occur near the isoelectric point (small charge …)
– the process can be followed photometrically (at 580 nm for Au NPs)
coagulation can be initiated by e.g. addition of sodium chloride –
change of ionic strength
Nanogold (colloidal gold)
a suspension / colloid of gold in a fluid
10 nm particles absorb green light and thus appear red
the size goes down, the melting temperature decreases
gold ceases to be noble
turn into insulators
shape: icosahedral symmetry, or hollow or planar,
depending on size
Usage of nanogold
colloidal gold is widely-used contrast agents for biological
electron microscopy
Au NPs can be attached to many traditional biological probes
such as antibodies, lectins, superantigens, glycans, nucleic
acids, and receptors
“immunogold” - antibody labeled with AuNPs, is used since 1970
particles of different sizes are easily distinguishable in electron
micrographs, allowing simultaneous multiple-labelling
experiments
colloidal gold has been successfully used as a therapy for
rheumatoid arthritis in rats; implantation of gold beads near
arthritic hip joints in dogs has been found to relieve pain
combination of microwave radiation and colloidal gold can
destroy the beta-amyloid fibrils and plaque which are associated
with Alzheimer's disease
possibilities for numerous similar radiative applications are also
currently explored
AuNPs are being investigated as carriers for drugs such as
Paclitaxel
nanosized particles are particularly efficient in evading the
reticuloendothelial system
in cancer research, colloidal gold can be used to target tumors
Nanogold / medical apps
Preparation
monodispersed NPs are more useful for practical use
reduction of tetrachloroauric acid HAuCl4 using different reagents:
sodium citrate - 15 to 150 nm large NPs, according to concentrations
sodium ascorbate - moderate 6 to 15 nm NPs
smallest NPs < 5 nm – using white phosphorus
sodium borohydride ~ 2 nm
structure of the coloid Au NP
coated with negative [AuCl2]prevents
coagulation
Reduction and size of NPs
for transition metals - the stronger the reduction reagent, the smaller
the produced nanoparticles
stronger reduction reagent would generate an abrupt surge of the
concentration of growth species
resulting in a very high supersaturation
and a large number of the formed initial nuclei
for a given concentration of metal precursor, the formation of a large
number of initial nuclei would result in a smaller size of the grown
nanoparticles
A
Effect of polymer
strong absorption of polymer would occupy the
growth sites and reduce the growth rate of
nanoparticles
full coverage of polymer can hinder the diffusion of
growth species from the surrounding solution to the
surface of the particles
in addition, polymer may interact with solute, catalyst,
or solvent and affect reaction
Effect of the shape of NPs
absorption spectra reflect also the shape of NPs
for Au nanorods, another resonance maximum appears
position depends on the length / diameter ratio
A
Enhanced
sensitivity
for assays / imaging applications,
a higher sensitivity is achieved by
increasing the size of NPs
catalytic deposition of silver
– increased size of NPs in the course of
the assay
– specifically bound Au NPs function
as nucleation sites for further growth
of crystals in the presence of Ag+ salts
Adsorption of proteins
incubation with protein, centrifugation, resuspension
Au NPs with protein A (G, L) – universal labeling of
immunocomplexes
– adsorption at pH 6,9 with polyethylenglycol (PEG 20 kDa, 0,025%) as
the stabiliser
– conjugate kept in 10 mM phosphate buffer pH 7.4 containing 1% PEG
adsorption of antibodies
– pH 8 až 9, PEG can be substituted with albumin (0.25%)
other useful conjugates
– lectins – detection of saccharides decorating cellular surfaces
– avidine / streptavidine – universal use
rapid visual immunotests (strips)
– red colours is easy to detect, high sensitivity
– stability of NPs – no degradation, an advantage compared to biolabels
(enzymes, …)
– zero toxicity
Au NPs for microscopy
microtubules
– anti-tubuline Ab, then goat
Ab anti mouse IgG with
coloid gold (left) and
NANOGOLD (right)
– magnified 1300x
contrast enhancing agent
prostate adenocarcinom
– Ab anti cytokeratine
antibody, conjugate Alexa
Fluor 488 FluoroNanogold
with the Fab' fragment of
goat Ab anti mouse IgG
– left, fluorescence
– right, localized Au after
enhancement with Ag+
Imaging of cells
different binding of Au NPs depending on the state of cells
Nanoclasters of gold
in fact, these are coordination complexes
central atoms Au are in the given configuration, at the surface are Au atoms
coordinated with a suitable ligang – Au valency becomes saturated and thus
stabilised
products as Undecagold and Nanogold
– tris(aryl) phosphine
– halid anionts
smaller clasters as Hexagold (6 Au) and Octagold (8 Au) can be charged
surface ligands adapted for simple bioconjugation
maleimide – for coupling with sulfhydryl groups (-SH)
Optical properties of Au NPs
metal NPs were used for glass staining from the ancient times
late 17th century - combining aqua regia solution of gold and tin
produces a precipitate with deep and vibrant red color
“purple of Cassius”, the colorant became one of the most successful red
pigments used in the production of glass and ceramics
“ruby glass” - essentially glass containing gold nanoparticles
a classic example of an ancient piece of art gaining its appeal from the color
produced by metal nanoparticles is the late Roman “Lycurgus Cup” - extraordinary
dichroic behavior exhibiting red color in transmission and green color in reflection
due to absorption and scattering of Au and Ag NPs in the glass
Surface plasmon resonance (SPR)
… prominent spectroscopic feature of noble metal NPs
gives rise to a sharp and intense absorption band in visible range
physical origin is a collective resonant oscillation of the free
electrons of the conduction band of the metal
surface plasmon oscillations induced by
an oscillating electric field in a metal sphere.
The displacement of the conduction electrons
(green) relative to the nuclei (gray) is shown.
The frequency of SPR is denoted omega
for NP much smaller than wavelength of the
incident light, its response to the oscillating
electric field can be described by dipole approximation
of Mie theory
the wavelength-dependent extinction cross section of a single particle,
Cext(λ), defines the energy losses in the direction of propagation of the
incident light due to both scattering and absorption by the particle
described in terms of the dielectric function of metal, ε(λ) = ε'(λ) + iε(λ), and
the dielectric constant of the medium, εm
SPR of NPs cont.
Cext depends on the dielectric function of the metal of
which the particle is composed - different absorption and
scattering characteristics for different metal NPs
maximum of Cext(λ) - the resonance condition, will take
place when the denominator of the right-hand side of the
equation becomes minimal
approximately at the wavelength λp for which ε'(λp) = -
2εm, if the imaginary part of the metal dielectric function,
ε(λp) is small
the resonance condition implies that the SPR frequency
depends on dielectric constant of the medium, εm
it is possible to observe adsorbed layers – sensing
Effect of the size of NPs
molar extinction coefficient of gold nanoparticles increases
roughly cubically with the particle radius
size of NPs can be obtained from the absorbance spectra
very high extinction coefficients, also size-dependent
Localised SPR
LSPR sensing
binding of target molecules to
modified Au NPs results in shift in
the absorbance spectrum
Single NP LSPR sensing
placing a chromophore near a resonant metal nanoparticle
– effect of the near field:
electric field surrounding a
resonant nanoparticle
(E=Ez)
dye
layer
enhanced fluorescence
reduced decay times
Plasmon enhanced optical absorption
the increased absorption cross section is accompanied by a
decrease in fluorescence lifetime.
plasmon resonance results in local enhancement of the electric
field: doubling the electric field quadruples the light absorption.
a single dye
molecule is only
visible in
fluorescence
when the gold
NP passes over
it
enhanced fluorescence
Plasmon enhanced effects
Silver nanoparticles (AgNP)
size between 1 nm and 100 nm
while frequently described as being 'silver‘, some are
composed of a large percentage of silver oxide due to
their large ratio of surface to bulk silver atoms.
synthesis - different routes, physical vapor deposition,
ion implantation, and wet chemistry
monodisperse nanocrystals of silver nanocubes were
synthesized in large quantities by reducing silver
nitrate with ethylene glycol in the presence of
poly(vinylpyrrolidone) (PVP) at 160oC
t ≠160 … irregular shapes
[Ag+] < 0.1 M … nanowires
Medical uses of Ag NPs
There is an effort to incorporate silver nanoparticles into a wide range of
medical devices
bone cement,
surgical instruments,
surgical masks,
wound dressings
treatment of HIV-1.
Samsung has created and marketed a material called Silver Nano, that
includes silver nanoparticles on the surfaces of household appliances
Silver nanoparticles have been used as the cathode in a silver-oxide
battery
some local research institute considered addition of silver NPs to fibers
for underwear (socks …)
Magnetic nanoparticles
xx
Magnetic field
permanent magnet has two poles forming the
magnetic dipole
– reprezented by an arrow in the S->N direction
– magnetic field exists between the poles
magn. dipole m in the magnetic field (gradient B)
is twisted by a torsion force to be parallel with the
magn. field force lines
magnetic materials have permanent magnetic
moment - magnetisation M, this depends on:
– density of magn. dipoles per unit of volume of the material
– intensity and mutual orientation of dipoles
– is due to the non-paired spins of electrons, little bit also due to the movement of
electrons within the orbital
magn. field can be realised by electromagnet
– electric current flowing through a conductive coil
(solenoid) forms inside a homogeneous field
density of flow B depends on the intensity H
and permeability of vacuum µ0: )(0
MHB += µ
Magnetisation
magnetisation M changes in dependence on the intensity of the
magn. field H
– coefficient is magnetic susceptibility χ
– size of the change depends on the type of material,
temperature and sometimes also on the “history”
of the previous magn. field (hysteresis)
heating of the magentised material decreases its magnetisation M,
above the critical temperature Tc (Curie temperature) magnetisation
M completely dissapears
H
M=χ
Magnetic materials
magnetic – exhibit tendency to “concentrate"
force lines; magnetisation permanent even in the
absence of magn. field
– Fe, Co
diamagnetic – force lines are slightly repelled
– proteins, fats, water
– small negative magn. susceptibility, Larmor diamagnetism
paramagnetic
– in magn. field their internal magn. dipoles become oriented and the
material gains magnetisation; Pauli paramagnetism
superparamagnetic
– typically nanoparticles 1 to 10 nm
– become oriented in the magn. field
exchange (electron-electron) interaction (many-particle wavefunction
antisymmetry)
– atomic scales
dipole-dipole interactions between locally ordered magnetic regions
– dipole interaction energy grows with the volume of the ordered region
– size of individual domains is set by a competition between volume and surface energy effects
– hundreds of atoms to micron scales
magnetic anisotropy energy
– magnetization interacts with angular momentum of the atoms in the crystal
– many microns
Magnetic interactions
Super-paramagnetic particles
ferromagnetic domains, created by d-electrons exchange interactions,
develop only when a cluster of iron atoms reaches a critical size (ca. um)
the magnetic moment per atom decreases toward the bulk value as cluster
size is increased
stable domains cannot be established in crystals that are smaller than the
intrinsic domain size
small particles can have very high
magnetic susceptibility with
permanent magnetic dipole
small clusters consisting of a single
ferromagnetic domain follow the
applied field freely
- superparamagnetism
magnetic susceptibility of
superparamagnetic particles is
orders of magnitude larger than
bulk paramagnetic materials
magnetic hard drives are based on a nanostructured device, called
giant magnetoresistance sensor
Albert Fert, Peter Grünbers Nobel Prize in Physics 2007
Hitachi hard drive reading head
Co, magnetic layer
NiFe alloy, magnetic layer
an easily re-alignable magnetization
Cu, electrically conducting layerlayers have a width that is
smaller than electron
scattering length
magnetization on the surface of the
disk can be read out as fluctuations
in the resistance of the conducting
layer
Giant Magnetoresistance
for antiparallel magnetic layers both spin polarizations are
scattered, giving rise to super-resistance (II)
data storage systems
giant magnetoresistance occurs when the magnetic layers above
and below the conductor are magnetized in opposite direction
electron scattering in magnetic
media is strongly dependent on
spin polarization.
when magnetic layers are
parallely magnetized, only one
spin polarization is scattered
(I,III).I II III
Magnetic NPs
wide range of applications:
– immunoassays
– separation of proteins and cells
– drug, oligonucleotides, … delivery
– magnetic hypertermia
– magnetic labels
– magnetic resonance imaging (MRI)
superparamagnetic materials based on
maghemite (γ-Fe2O3) and magnetite (Fe3O4)
– good magnetic properties, low toxicity
– required properties: uniform shape, defined crystalinity,
monodispersity, stability in water, surface reactive groups
– NPs or hollow spheres (higher magn. moment)
Preparation methods
deposition from gas phase
termal decomposition
– Fe(CO)5, Fe(oleate)2, Fe tris(acetylacetonate)
microemulsion precipitation
sonochemical synthesis
hydrolytic reactions
– heating up ferric chloride FeCl3.6H2O with hexamethylenediamine and sodium
acetate in glycol (6 hours, 200 oC)
– resulting particles provide surface aminogroups -NH2
bacterial (BMP) production
– NPs coated with phospholipids layer
(magnetosomes)
surface functionalisation
– thin layer of gold (core-shell, also protective)
– polymer coating with functional groups
– silanisation using aminopropyltriethoxysilanu
(APTES)
– electrostatic adhesion
(over polyamine coating)
100 nm
Magn. particles in liquid
effect of the magn. field
– uniform field orients the particle according to its magn. moment, but no
translational movement occurres
– for translation, the gradient of magn. field is required
viscosity effects play significant role
magnetic and electrostatic forces among particles
– might result in aggregation and even precipitation
– electrical repulsion prevents this undesired process – appropriate surface
charge
Separation of cells
direct method
– ligand immobilised at the surface of magn. NPs (up to 50 nm – otherwise
mechanically stressful for the cells)
– added to solution, specifically bound to the surface of target cells
indirect method
– target cells are labeled with ligand – e.g. antibody (biotinylated …)
– excess of the label is washed away
– labeled cells are specifically bound to modified magnetic particles (with
streptavidine …)
the resulting complex is easily isolated with magnetic separator
– a suitable permanent magnet
could be either positive or negative isolation
– separated are either target or balast cells
Magnetic sorting of cells labeled
with superparamagnetic beads
HM χ=
z
B
MF zz
∂
∂
=
)(0 MHB += µ
Induced magnetic moment:
Magnetic force:
Particle were pulled to point of highest field gradient
Copyright Stuart Lindsay 2009
MFS: microfabricated
ferromagnetic strips
Super-paramagnetic separations
Delivery (magnetic targeting)
accumulation of active substances (drugs, …) in the chosen target
part of the organism
– injection to the blood vessel supplying the relevant organ
in the presence of external magn. field
– should overcome local linear flows (0.05 to 10 cm/s, according to diameter and
network of blood vessels)
– long-term accumulation in the chosen place (~ 70%)
– local high concentration of the released compound (up to 8x)
– significantly lower complications for other organs when compared to the
system-wide application
thermosensitive magnetoliposomes
– in the target site, the delivered substance is released by local heating using the
magnetic field (alternating – mechanical movement of particles – heat
generation)
Hyperthermy using magnetic fluids
damage of tumors using locally increased temperature (42 to 46 oC)
– decreased viability of cells in the tumor, these become more sensitive towards
chemo / ratiotherapy, magn. NPs selectively bound to malignant cells
Contrast bioimaging
MRI, "magnetic resonance imaging" - H-NMR applied on tissues
for MRI, contrast is due to different responses of individual tissues
on the applied radiofrequency pulses
– density of protons and magn. relaxation times corresponding to the chemical
composition, mainly content of water and lipids
superparamagnetic NPs (magnetite-dextran) in the target site greatly
improve the contrast
– more NPs – darker image, effect on the rate of relaxation of protons from the
excited state
– healthy cells accept NPs, damaged or dead cells do not take NPs
internal cell label – monitoring of cells added to the organism within
cell-based therapies
– bone marrow cells tranplantation, stem cells
SPIO
superparamagnetic iron
oxide .. NPs
Bioanalytical applications
immunomagnetic sensors – simple regeneration of the sensing
surface
– at the surface of the transducer, magn. particles with immobilized
biorecognition element (antibodies, …) are attached with the help of magnet
/ electromagnet
– after completion of the immunoassay, the consumed particles are replaced
with fresh ones
– vhodné zejména pro komplexní vzorky (potraviny, krev), které normálně
degradují imunorekogniční vrstvu
efficient preconcentration of the target analyte from complex
samples
– simple washing and clean-up procedures
enzyme biosensors – regeneration for inhibition-based assays
analysis of DNA
– specific extraction of the target sequence from complex sample matrix