Fluorescent nanoparticles
from quantum dots to up
up--conversion:
conversion: preparation,
preparation,
purification,, bioconjugation and applications
purification
Petr Skládal
Skládal
Department of Biochemistry,
Biochemistry, Faculty of Science
RG & CF Nanobiotechnology,
Nanobiotechnology, CEITEC
Masaryk University, Brno
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Nanobiotechnology at MU
we have started research in this area around 2005
(purchase of our first AFM Ntegra Vita at NCBR)
significant expansion within CEITEC – Structural
Biology research program – the Core Facility of
Nanobiotechnology
equipment: AFM – Bruker FastScanBio, JPK
Nanowizard3 + ForceRobot, NTMDT SolverNext,
Ntegra Vita; ink-jet deposition FlexArrayer S3,
fluorescence chip reader InnoScan 1100
research: biosensors, atomic force microscopy for
bio, nanoparticles
team: Petr Skládal, Jan Přibyl, Antonín Hlaváček,
Karel Lacina + 11 PhD students, 15 MSc, Bc students
Nanoparticles (NP)
nano is the SI prefix of the multiples of 10-9
derived from the Greek νᾶνος – dwarf
NPs are particles with a size from 1 to 100 nm
larger than small molecules, but the polymers (proteins, nucleic
acids, dendrimers) generally not included
polymeric NPs and covalent nanocrystals (diamond, silicon, quantum
dots...) are commonly regarded as NPs
liposomes and polymersomes are usually not marked as
nanoparticles, but rather refered as a "nanovesicles”
alternative definition: nanoparticles is object, whose one dimension
is in the range from 1 to 100 (1000) nm; however, it is probably
preferable to mark these structures as nanofibers and nanoplates
size range 1-100 nm overlaps with sizes of colloidal particles (1 to
1000 nm) and mesoscale; therefore, it should be distiguished
between NPs and colloidal particles
NPs are usually used as a dispersion in aqueous solutions
Preparation of NPs
in solution, A + B → C(s)
product C is insoluble, it forms nucleation centers
which subsequently increase size resulting in NPs
advantages:
– simple control of conditions (c, p, t)
– instrumentation not complex
– often result monodisperse NPs
problems
– protocols are mostly empirical
– numerous steps – preparation of mixture, reaction, washing,
drying, purification, …
quantum dots
upconverting NPs
Fluorescence
Fluorescen
ce
intensity I0
fluorescence
light source
c
fluorescence = photoluminiscence
photoluminiscence,,
emission
emis
sion of photon after return of electron
from a higher energy level, where it was
I0 >> If
transfered by photoexcitation
fluorescence F is measured at a given wavelength
If
D
detector
I = kΦ εcl
– emission x excitation spectra
f
– relative units, also as a count of photons per unit area
– other parameters: quantum yield Φ, mean lifetime τ, polarisation P
fluorescence typically ends immediatelly (10-8 s) after the end of
excitation
phos
phosph
phorescence
orescence is longer
longer,, excita
excitation
tion provides metastable states
energy transfers - Jablonsk
Jablonskii diagram
diagram::
Fluorescence – Jablonski diagram
relaxation
relaxa
tion
S2
internal
transfer
inter--system transfers
inter
S1
T1
external
transfer
absorbtion
absorb
tion
non-radiation
nonsteps
absorbtion
absorbtion
colision
vibrational
levels
S0
S0, S1, … energy
energy levels
τ≈
10-15 s
fluorescence
ph
phos
osph
phorescence
orescence
10-8 s
10-3 - 1 s
Quantum dots (QDs)
fluorescent semiconductor nanocrystals
– size 2 - 20 nm
nm,, materials as CdTe, CdSe, PbS, InP, Si
– often core / shell type: CdSe / ZnS, InP / ZnS,
ZnS, …
quantum properties between semiconductors and
isolated atoms
– semiconductors – valence and conductive bands separated by Eg
– excitation - electron moves to the conductive band, leaves
positive hole in the valence band, spatial separation of this pair
("exciton") corresponds to the Bohr diameter, typically 1 to 10 nm
QD
QDs
s have similar size
– excitons bound similarly as a
“particle in the box”
– energetic levels similar to atoms
or molecules
– optical properties depend on the
size (and material, …) of QDs
Interesting optical properties
spe
spec
ctr
tral
al properties are “tunable” by size changes
– e.g. CdSe nanocrystals can emit from 450 to 650 nm
– smaller QDs are blue (hypsochromic effect)
advantages of QDs (vs. classic fluorophores)
– high brightness (20x higher, high quantum yield, high extinction
coefficients)
– narrow emission bands (< 30 nm, large Stokes shifts –
multiplexing, coding, …)
– high stability, low photodegradation, resist photooxidation
(polymeric / silicate outer shells)
– wide absorbtion bands in near UV – common excitation source
Colors vs. materials / sizes
QDs: absorbtion / emission spectra
QDs
QD
s … synthesis
CdTe core with CdS shell
coating with thioglycolic
thioglycolic acid (TGA)
prec
pre
cur
ursors
sors - Te, CdCl2, TGA and NaBH4
– NaHTe produced by reduction of Te using NaHB4
– solution of NaHTe injected to the mixture of CdCl2 and TGA
under inert atmosphere (argon)
– heating under reflux (minutes … hours)
during heating, the size of QDs gradually increases
larger QD exhibit a better stability
Experiments with QDs
QDs
QDs may be toxic
CdSe and CdTe easily prepared in water, most common in
bioapplications
– high quantum yields (~50%), after conjugation decreased (~20%)
“classic” materials - CdTe
CdTe,, CdSe
CdSe,, HgTe
HgTe,, PbS ... are toxic
novel options – Si
Si,, ZnS dop
doped
ed with Mn
Mn,, InP
InP,, InP / ZnS,
ZnS, Ag2S, Ag2Se,
Se, C
dots
carbon
carbon--based QDs (C(C-dots,
including Si doped)
formed during decomposition of
organic substances
– carbon black from nanotubes,
paraffine wax
– emission spectra depend on size and
excitation wavelength
Multiplexed immunoassays
different QD-based labels, determination of several
analytes (markers) during a single procedure
Bar--coding of (nano
Bar
(nano/micro)
/micro) particles
optical code based on mixtures
of several fluorescent QDs
characteristic spectra
large--scale reactions carried out
large
in a single tube
code – option to identify the
successful products
combinatorial methods
Bar coding
Capillary flow multiplex assays
excitation
code – type of the biorecognition element on the bead
common secondary label – quantification (or indication of presence)
suitable for antibody / antigen, oligonucleotides, …
Anti-Stokes fluorescence
organic fluorophores – excitation with shorter wavelength, emission
at a longer wavelength, intensity of emission is directly proportional
to the excitation
however, a longer wavelength can be changed to a shorter one –
energy of few photons is summarised and a higher energy photon
becomes emited; intensity depends on the square of excitation –
non-linear processes
two(m
two(multi
ulti))-photon luminescence
– several photons interacting with a single molecule
– e.g. 2-photon confocal microscopy – lower excited volume, better resolution
second harmonic generation
– increased energy of photons passing through suitable crystalic materials
– double frequency at the output, narrow emission peaks, coherence
– nanocrystals of KNbO3, LiNbO3, BaTiO3, ZnO
„upconvertion
pconvertion““
Upconverting NPs (UCNP)
gradual absorbtion of several photons
lower excitation intensity compared to
multiphoton techniques
– observed for materials doped with Ti, Ni, Mo, actinoids
quite high for NPs based on NaYF4 doped with
lanthanoids (eg. Yb3+ and Er3+, or Yb3+ and Tm3+)
Mechanism of upconversion
ESA (excited
(excited--state absorption
absorption))
– ions gradually absorb several photons
– transfer to the target band, emission starts
ETU (energy
(energy transfer upconversion
upconversion))
– ions with a higher extinction coef.
(sensitizers) become excited by
absorbtion of photon and transfer
energy to nearby ions (emitters)
beta phase of NaYF4 doped with
Yb3+ (20%, sensitiser) and Er3+, (2%, emitter)
Assays with UCNP - LRET
detection of human IgG
NaYF4 : Yb : Er
overlap of Au absorbtion with
green emission (energy
transfer)
homogeneous competition
UCNPs
Au NPs
NPs (for bioapplications) in solution
protective / functional outer layer is required
chemical stabilisation of the surface
– Van der Waals, electrostatic, solvatation, hydrophobic and steric
interactions
stabilisation of the NPs dispersion
– interactions with solvents, among NPs, with surfaces
– repulsive and attractive; the latter result in aggregation of NPs,
adsorption on surfaces – undesired
– can be controlled by modification of NPs surface
– core / shell components
reactive groups suitable for coupling of bioligands
Nanobioconjugates
NPs are similar to biomolecules in size, which enables creation of
nanostructures bearing biological function of the biomolecule
– specific interaction of antibodies with antigen and interesting properties
of NPs, which facilitate their detection luminescence, catalysis, …
such structures are called bioconjugates of
NPs (nanobioconjugates) and have wide application in biology,
medicine and analytical chemistry
Coupling reactions
Nanoparticles:
carboxyl –COOH
amine -NH2
thiol –SH
aldehyde -CH=O
epoxy group
maleimide group
saccharides
biotine
oligonucletides
bioorthogonal groups, click
chemistry
Bioligands:
carboxyl –COOH
amine -NH2
thiol –SH
aldehyde -CH=O
saccharides
avidine
oligonucletides
other introduced groups,
click chemistry
Maleimide + -SH
for reduced antibodies
Purification of NPs (NP conjugates)
isolation of just prepared NPs
Improved prooperties (separation by shape, size, ...)
removal of excess modifying agents (surface ligands,
small molecules introducing functional groups) or
biomolecules in the preparation of bioconjugates
purification of bioconjugates
precipitation
extraction
dialysis
chromatography
filtration
centrifugation
electrophoresis / isotachoforesis
Size-exclusion chromatography
separation of QD-protein
conjugates
Hlavacek, A., Bouchal, P., Skládal, P. (2012) Microchim. Acta 176, 287-293
Electrophoresis
in agarose gel,
separation is more dependent on the size of NPs
bioconjugates – shift of mobility
Isotachophoresis
electric field produces sharply bounded zones containing
the separated nanoparticles
purification and concentration of nanoparticles
Hlavacek, A., Skládal, P. (2012) Electrophoresis 33, 1427
Aggregation of NPs?
gel elfo of UCNPs
Conclusions
we like most types of nanoparticles
experience in preparation of metal NPs, magnetic
SPIO, QDs, UCNPs
extended knowledge in bioconjugations,
characterisation and purification of
bioconjugates
know-how and NPs provided within Core Facility
– Nanobiotechnology at CEITEC MU
open access for academic users