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Markéta Vaculovičová
Laboratory of Bionalysis and Imaging
Nanoparticles in bioanalytical
applications
The size of Things
Definition of Nanotechnology
The name “nanomaterials” is a general term that covers an
extremely large group of materials. A generally accepted
definition is that a nanomaterial is “any material that has an
average particle size of between 1 and 100 nanometres
at least in one dimension.”
The European Commission defines a nanomaterial as
“a natural, incidental or manufactured material containing
particles, in an unbound state or as an aggregate or as an
agglomerate and where, for 50% or more of the particles in
the number size distribution, one or more external
dimensions is in the size range 1 nm–100 nm”
× albumin molecule is approximately 7 nm in diameter
Richard Feynman’s famous presentation “There’s Plenty of Room at
the Bottom” was in the 1959 at the American Physical Society.
Here he asked:
•Why can’t we manipulate materials atom by atom?
• Why can’t we control the synthesis of individual molecules?
•Why can’t we write all of human knowledge on the head of a pin?
•Why can’t we build machines to accomplish these things?
The challenge involved the possibility of scaling down letters small enough so as to
be able to fit the entire Encyclopedia Britannica on the head of a pin, by writing
the information from a book page on a surface 1/25,000 smaller in linear scale
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What is nanotechnology, and why do we care? What is nanotechnology, and why do we care?
nanotechnology is the study of matter from 1-100 nm
What is nanotechnology, and why do we care?
nanotechnology is the study of matter from 1-100 nm
for chemists: the interface between molecules and material
What is nanotechnology, and why do we care?
nanotechnology is the study of matter from 1-100 nm
for chemists: the interface between molecules and material
for physicists: where quantum ends and bulk begins
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What is nanotechnology, and why do we care?
nanotechnology is the study of matter from 1-100 nm
for chemists: the interface between molecules and material
for physicists: where quantum ends and bulk begins
for you: faster computers, better communication, and
new approaches to medicine
Interdisciplinary area :
Biology, Physics, Chemistry, Material science, Electronics,
Chemical Engineering, Information technology
Why Now?
• New tools for atomic-scale characterization
• New capabilities for single atom/molecule
manipulation
• Computational access to large systems of
atoms and long time scales
• Convergence of scientific-disciplines at the
nanoscale
What’s the BIG deal about
something so SMALL?
Materials behave differently at this size
scale.
It’s not just about miniaturization.
At this scale---it’s all about INTERFACES
Color depends on particle size
Quantum dots 3.2 nm in diameter have blue emission
Quantum dots 5 nm in diameter have red emission
Carbon particles
• Carbon nanotubes
• Graphene
• Fullerenes
Polymer particles
• Silica
• Chitosan
• Polystyrene
• Dendrimers
Metal particles
• Noble metals
• Iron oxide
• Semiconductors
Bioparticles
• Liposomes
Particle types
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 Semiconductor nanocrystals synthetized from II and VI
or III and V elements of PSE
 1970 developed first low dimensional structures
quantum well (QW)
 1980 – Ekimov, Efros – first description of quantum
dots
Quantum dots
Nanoparticles for sample
pretreatment
Sample preparation
• Isolation form complex biological
matrixes
– Large surface
– Simple functionalization
– Magnetic properties
• Magnetic nanoparticles (Iron oxides),
Carbon nanoparticles (nanotubes,
graphene), gold nanoparticles
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CE characterization of
nanoparticles
Li, Analytica Chimica Acta 647 (2009) 219–225
Separation of Quantum dots
Electropherograms of a mixture of four QDs. Sample were water-soluble CdSe/ZnS
core–shell QDs with emission wavelength at 534, 580, 618 and 691 nm, and
concentrations of QDs in the mixture were all 2.038×10−6 M. 4% PEG was used as
sieving medium and applied voltage was 14 kV
Separation of latex nanoparticles
separation of sulphated polystyrene latex particles.
BGE: 89 mmol/L Tris,
89 mmol/L boric acid, 2 mmol/L Na2EDTA, pH 8.3
Radko, S. P., Stastna, M., Chrambach, A., Electrophoresis 2000, 21, 3583–3592.
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Separation
Covalently
bound
opentubular phase
3
2
A B
4 A B C
A B
1
A B
C D
Scanning electron micrographs of various nanomaterials immobilized on the capillary wall.
1) Scanning electron micrographs images of prepared CNTs-polymer composites. (A) Mono(2-(methacryloyloxy)ethyl)
succinate carbon nanotubes (MES-CNTs) coated on capillary
wall; (B) five-fold magnification of (A); (C) the MES-CNTs formed on aluminum foil; (D) butyl
methacrylate carbon nanotubes (BMA-CNTs) coated on capillary wall. 2) SEM images of (A)
the BMA-CNTs composite, and (B) as a coating on a capillary wall 3) Scanning electron
micrographs of (A) bare fused-silica capillary column, and (B) capillary column modified with
octadecylamine-capped AuNPs. 4) Scanning electron micrographs of TiO2 NPs-coated
column. (A) Cross section of the column prepared by two-cycle coating procedures. (B)
Edge of the column prepared by two-cycle coating procedures. (C) Edge of the column
prepared by a single coating procedure.
A B
C D
E F
Separation of 100 bp DNA step ladder using (a)
0.10% PEO only and (b) 0.02% yttrium oxide
nanoparticles and 0.10% PEO, peak
assignment; 1 = 100 bp. 2 = 200 bp. 3 = 300 bp. 4 =
400 bp. 5 = 500 bp. 6 = 600 bp. 7 = 700 bp. 8 = 800 bp.
9 = 900 bp. 10 = 1000 bp. 11 = 1500 bp.
Separation of 500 bp DNA ladder using (c)
0.02% PEO only and (d) 0.02% yttrium oxide
nanoparticles and 0.02% PEO,
Separation of 1 kbp DNA ladder using (e)
0.02% PEO only and (f) 0.02% YNP and 0.02%
PEO,
peak assignment; 1 = 1000 bp. 2 = 2000 bp. 3 = 3000
bp. 4 = 4000 bp. 5 = 5000 bp. 6 = 6000 bp. 7 = 7000
bp. 8 = 8000 bp. 9 = 9000 bp.
Electrokinetic injection at 4 kV for 3s; separation at 5.4
kV, fused-silica capillary; 360 μm o.d., 75 μm i.d.,
30 cm total length, and 22 cm effective length.
Kwon, H.; Kim, Y. Bull. Korean Chem. Soc. 2009, 30, 297.
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Cao, J.; Dun, W. L.; Qu, H. B. Electrophoresis 2011, 32,
408.
Influence of SC-SWNTs concentrations on
the separation of eight analytes using
MEEKC: (A) without SC-SWNTs, (B) 1.5
mg/L SC-SWNTs, (C) 3.0 mg/L SC-SWNTs,
(D) 4.5 mg/L SC-SWNTs, and (E) 6.0 mg/L
SC-SWNTs.
Microemulsion buffers: 0.5% (57 mM) w/v ethyl
acetate, 0.6% (30 mM) w/v lauric acid, 4.0% (666
mM) w/v propanol, 50 mM Tris solution of pH 9.0
and 0–6.0 mg/L SC-SWNTs additives. Other
operating conditions: analyte concentration, 0.07
mg/mL; capillary length, 40 cm total (31.5 cm
effective length) 50 μm id; temperature, 30°C;
voltage, 23 kV; injection, 50 mbar, 3 s; detection,
200 nm; analytes: 1. epicatechin, 2.
epigallocatechin gallate, 3. epicatechin gallate, 4.
Caffeic acid, 5. Gallic acid, 6. Protocatechuic acid,
7. Quercetin, and 8. Kaempferol
In-capillary magnetic isolation
4.5 5.5 6.5
0.02
4.5 5.5 6.5
0.3-
4.5 5.5 6.5
0.5Migration
time (min)
Fluorescenceintensity(a.u.)
●
○
●
○
●
○
0.08 µM
miRNA
0.63
µM
miRNA
5.00
µM
miRNA
27Bioanalýza - separace
In-capillary magnetická izolace
Detection
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Nanomaterials in electrochemical detection
Nanoparticle modified electrodes
Nanoelectrodes
High surface → high adsorption capacity →
increased sensitivity
Carbon nanotubes, Au and Pt nanowires
Analyte Detection
1
3
2
54
A
B
A B
C D
A
B
A
B
A
B
Scanning electron micrographs of
various nanomaterial-modified electrode
surfaces used for electrochemical
detection in CE. 1) SEM micrographs of
bare SPE electrode (A), and CNTs
dispersed on the SPE electrode surface
(B), 2) SEM images of the surfaces of
A) pure PMMA sheet, B) pure CNTs,
and the cross sections of C)
graphite/PMMA and D) CNT/PMMA
composites. 3) SEM pictures of carbon
nanotube paste electrode prepared with
short (A) and long (B) carbon
nanotubes, 4) A) SEM image, and B)
energy dispersive spectroscopy results
for the CNT–alginate composite on a
carbon disk electrode. 5) SEM images
of pristine graphene (A), and
graphene/poly(urea-formaldehyde) (B)
composite.
Electropherograms for hydrazines (A), dopamine, catechol, and ascorbic acid (B), phenols (C), and
purines (D) at the bare (a) and CNT-modified (b) screen-printed carbon electrodes.
Sample A: 100 μM hydrazine (1) and 200 μM dimethylhydrazine (2).
Sample B: 100 μM dopamine (1), 100 μM catechol (2), and 100 μM ascorbic acid (3).
Sample C: 100 μM phenol (1), 100 μM 2-chlorophenol (2), 200 μM 2,4-dichlorophenol (3), and 200 μM
2,3-dichlorophenol (4).
Sample D: 200 μM guanine (1) and 200 μM xanthine (2).
Conditions: run buffer, phosphate buffer (20 mM, pH 7.5) (A); 20 mM MES (pH 6.5) (B); 10 mM
borate/20 mM phosphate buffer (pH 8.0) (C); 5 mM borate/10 mM phosphate buffer (pH 8.0) (D).
Separation voltage, +1000 (A) + 1500 (B-D) V; injection voltage, +1000 (A) +1500 (B, C), +2000 (D) V;
detection potential, +0.6 (A), +0.7 (B), +0.9 (C), and +0.8 V (D) (vs Ag/AgCl wire).
Wang, J.; Chen, G.; Chatrathi, M. P.; Musameh, M. Anal. Chem. 2004, 76, 298. Crevillen, A. G., Avila, M., Pumera, M., Gonzalez, M. C., Escarpa, A., Anal. Chem. 2007, 79, 7408–7415.
Representative examples of analytical
performance of CNT-film detectors in
microchip electrophoresis
(a) Bare SPE
(b) SPE–SWCNT,
(c) SPE–MWCNT-A
(d) SPE–MWCNT-B
(A) antioxidant standard
peaks: (1) arbutin, (2) phloridzin, (3)
catechin, (4) rutin, (5) ascorbic acid
(B) flavor standard peaks: (1) vanillic alcohol,
(2) ethyl maltol, (3) maltol, (4) ethyl
vanillin, (5) vanillin
(C) water-soluble vitamin standard peaks: (1)
pyridoxine, (2) vitamin C, (3) folic acid.
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Nanomaterials in optical detection
Nanoparticles as labels for fluorescent labeling
Nanoparticles for colorimetric detection
(i.e. pregnancy test)
Simple surface modification → applicable
for different analytes
Quantum dots (semiconductor, carbon)
Gold nanoparticles
Analyte Detection
IgG
HWR-QD
L. Janu, M. Stanisavljevic, S. Krizkova, P. Sobrova, M. Vaculovicova, R. Kizek, V. Adam, Electrophoretic study of peptidemediated
quantum dot-human immunoglobulin bioconjugation, Electrophoresis 34 (2013) 2725-2732.
Nanomaterials in elemental detection
Nanoparticles as labels for labeling
Simple surface modification → applicable
for different analytes
Quantum dots (semiconductor, carbon)
Gold nanoparticles
Analyte Detection
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Laser ablation ICP MS
Carbon Gold
IgG
Au NP
Dot blot
Tissue section
Imaging
BLI - bioluminescence imaging; CT - computed tomography; DOT - diffuse optical tomography; FMT - fluorescence-mediated tomography;
FPT - fluorescence protein tomography; FRI - fluorescence reflectance imaging; LN-MRI - lymphotropic nanoparticle-enhanced MRI;
MPM - multiphoton microscopy; MRI - magnetic resonance imaging; MSCT - multislice CT; OCT - optical coherence tomography;
OFDI - optical frequency-domain imaging; PET - positron-emission tomography
PET
CT
IMAGING
TECHNIQUES
• Au nanoparticles
• Microbubbles
• Magnetic particles
• Quantum dots
• Upconversion nanoparticles
• Radiolabeled
nanoparticles
(64Cu, 18F, Ga, Tc)
Advantages:
Real-time
Low cost
Disadvantages:
Low resolution
Operator dependent
Advantages:
High sensitivity
Quantitative
Whole-body scaning
No tissue penetrating
limit
Disadvantages:
Radiation risk
High cost
Advantages:
High spacial resolution
No tissue penetrating limit
Disadvantages:
Relatively low sensitivity
High cost
Long imaging time
Advantages:
High sensitivity
Multicolor imaging
Acivable
Disadvantages:
Low spacial resolution
Poor tissue penetration
Advantages:
High spacial resolution
No tissue penetrating
limit
Disadvantages:
Radiation risk
High cost
Ultrasound
Optical
Magnetic resonance
• Au nanoparticles
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Multimodal imaging
MR +
Optical
MR + PET
Magnetic
particle
Magnetic
particle
QD
Theranostics
Therapy + Diagnostics = Theranostics
drugnanoparticle
targeting
agent
imaging
agent
PET + Optical
Carrier
QD
Iron oxide (IO) particles functionalized with aminoterminal
fragment (ATF)
peptides labeled with near infrared (NIR) dye (Cy5.5)
enhance permeability and retention (EPR) effect
“Leaky vessels” – solid tumor, myocardial infarction…
Wide inter-endothelial junctions, fenestrae, transendothelial channels,
discontinuous basement membrane
Pore size (tumor):
200 nm-1.2 μm
380-780 nm a typical value
Pore size (normal vessels):
6-7 nm
Passive targeting
Peter Carmeliet. Oncology 2005;69(suppl 3):4–10
Active targeting
(B) Sagittal plane displaying the location of the 9L
xenograft tumor. (C) non-targeting PEG-coated
iron oxide nanoparticles and (D) CTX-targeted
PEG-coated iron oxide nanoparticles 3 h post
nanoparticle injection.
• Antibody-antigen
• Ligand-receptor
• Lectin-carbohyrdrate
• Aptamers
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Future prospective
• To meet the requirements for nanotheranostic agents, nanoparticles must be
developed with high stability in extreme conditions (high salt concentrations and
wide pH and temperature ranges) and retain minimum active interaction with
serum proteins, which would allow the nanoparticles to conjugate alternative or
additional biomolecules without substantially alternating its colloidal stability.
• The biocompatibility and toxicity of theranostic nanoparticles need to be
thoroughly evaluated as addition of each component material would potentially
alter the pharmacokinetic profiles of the nanoparticle.
• To further identify new molecular targets that are fully correlated with diseases
and discover new targeting ligands with high specificity, small molecular footprint,
and good stability.
• Future improvement will also need to focus on development of innovative
strategies to allow efficient tissue penetration of nanoparticles and offer
controlled delivery of therapeutics to a particular type of tissue or subcellular
compartments
Fluorescence imaging
http://research.stowers-institute.org/wiw/external/Technology/NLO/index.htm
0
1000
2000
3000
4000
5000
6000
0 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 24
h
Fluorescenceintensity[a.u]
CdTe-PVP QDs (ʎex 480 nm, ʎem 535 nm). Fluoresence intensity was analyzed in 3 leaf regions
(a-c) of the main venation as illustrated in the picture (left). Graph (right) shows the mean
fluorescence intensity in these regions.
0 h
Fluorescent imaging of plants
(a) Micrograph of PC3 cells that had been cultured in Ham's F12 medium for 5 days and stained
with GdQDs. The red fluorescent spots indicate the presence of damage on the plasma membrane
caused by the old medium, in which most of the nutrients had already been consumed by the cells.
Micrographs of PC3 cells treated with carboplatin (b) or cisplatin (c) and subsequently stained with
GdQDs. Different amounts of red fluorescence indicate different effects of the chemotherapeutic
drugs on the plasma membrane.
Fluorescent imaging of cells
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Pre-injection 1.5hour post-injection
injection
tumor
QDs injected into mouse with breast tumor
Fluorescent imaging of animals Fluorescent imaging of animals
Cleavable probes
• Enzymes
• Nucleic acids
• Reactive Oxygen Species
• pH
• Ions
• External stimuli (ultrasound,
temperature, light, etc.)
 Lower background signal
 Higher sensitivity
Summary
• Nanomaterials are great 
Analytical tools for characterization
of nanomaterials → Nanomaterials as
tools for improvement of analytical
methods
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Thank you for your
attention
http://ucb.af.mendelu.cz/ustav/laboratore/27216-laborator-bioanalyzy-a-zobrazovani
marketa.ryvolova@seznam.cz