1
MASARYK UNIVERSITY
FACULTY OF SCIENCE
DEPARTMENT OF CHEMISTRY
Capillary Electrophoresis:
A well-established method with a modern twist
Markéta Vaculovičová
Habilitation Thesis
Brno 2017
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Motto: “There are only two options, either it will work or it won’t “
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Acknowledgments:
At this place, I would like to thank to my husband Tom for his support and help.
I would like to thank also to Prof. Vojtech Adam for or the chance he gave me and for his
kind attitude during all these years.
Luckily, I got the chance to work with many brilliant coworkers, colleagues and students.
Above all I would like to name Prof. Jan Preisler and Prof. Mirek Macka. I have learnt a
lot from all of them.
I am grateful to all members of my group, my former and current students and my closest
associates for their contribution to the papers discussed in this work. Namely, I would like
to thank Luky Nejdl, Týnka Šmerková, Dave Hynek, and Simča Dostálová.
Last but not least, I am thankful to my family and friends.
Markéta Vaculovičová
Brno, November 2017
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Abstract:
This habilitation thesis is a commented collection of 11 selected peer-reviewed scientific
papers that deals with combination of nanomaterials with powerful and well-established
analytical method – capillary electrophoresis (CE). Such combination is beneficial for both
of these sides since CE is able to characterize properties of nanomaterials in terms of size,
charge or interaction with other compounds and on the other hand, nanomaterials are
applicable for improvement of CE performance both separation resolution as well as
detection sensitivity.
Abstrakt:
Tato habilitační práce je komentovaným souborem 11 vybraných recenzovaných publikací
zaměřených na kombinaci výhod nanomateriálů se schopnostmi vysoce účinné analytické
metody – kapilární elektroforézy (CE). Toto spojení oboustranně výhodné, protože CE
umožňuje charakterizovat nanomateriály z pohledu velikosti, náboje nebo interakcí
s jinými molekulami a na druhou stranu, nanomateriály lze využít pro zlepšení vlastností
CE jak z pohledu zlepšení separačního rozlišení tak zvýšení detekční citlivosti.
Key words: nanotechnologies, nanomaterials, nanoparticles, separation techniques
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Table of Contents:
1 INTRODUCTION ................................................................................................................................. 6
1.1 STRUCTURE OF THE THESIS ................................................................................................ 7
1.2 PERSONAL COMMENTARY .................................................................................................. 7
1.3 AUTHOR’S CONTRIBUTION ................................................................................................. 8
2 NANOTECHNOLOGIES, NANOMATERIALS AND NANOMEDICINE .................................. 12
2.1 NANOTECHNOLOGIES....................................................................................................... 12
2.2 NANOMATERIALS............................................................................................................. 12
2.2.1 Metal Nanoparticles........................................................................................................ 13
2.2.2 Semiconductor Nanocrystals........................................................................................... 14
2.2.3 Carbon nanomaterials..................................................................................................... 15
2.3 NANOMEDICINE ............................................................................................................... 16
3 CAPILLARY ELECTROPHORESIS IN NANOMEDICINE ........................................................ 19
3.1 CARGO ENCAPSULATION.................................................................................................. 19
3.2 CONJUGATION TO TARGETING LIGANDS........................................................................... 20
4 MONITORING OF THE INTERACTION BETWEEN NANOPARTICLES AND
BIOMOLECULES ........................................................................................................................................ 21
4.1 AFFINITY INTERACTION ................................................................................................... 21
4.2 NON-SPECIFIC INTERACTION ............................................................................................ 21
5 CE FOR NANOMATERIALS AND NANOMATERIALS FOR CE ............................................. 22
5.1 SAMPLE PRETREATMENT BY NANOMATERIALS ................................................................ 22
5.2 CHARACTERIZATION OF NANOPARTICLES BY CE ............................................................. 23
5.3 SEPARATION IMPROVEMENT ............................................................................................ 24
5.4 ENHANCEMENT OF DETECTION SENSITIVITY .................................................................... 25
6 IN-CAPILLARY QUANTUM DOT SYNTHESIS ........................................................................... 27
6.1 ORGANOMETALLIC SYNTHESIS OF QUANTUM DOTS.......................................................... 27
6.2 AQUEOUS SYNTHESIS OF QUANTUM DOTS ........................................................................ 27
6.3 THE OTHER WAYS OF SYNTHESIS...................................................................................... 28
6.4 UV IRRADIATION IN-CAPILLARY SYNTHESIS OF QUANTUM DOTS ..................................... 28
7 CONCLUSION .................................................................................................................................... 29
8 REFERENCES: ................................................................................................................................... 30
9 ARTICLES........................................................................................................................................... 35
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1 Introduction
Nanotechnology is a quickly developing area of science. Due to an outstanding lecture
given by Richard P. Feynman in 1959, giving the vision that science and technology can be
based on nanoscale, this year can be marked as turning-point in scientific history.
However, already Michael Faraday in 1857, observed characteristic behavior of gold
nanoparticles in aqueous solution. Yet, the oldest known application of nanomaterials
would probably be the creation of Lycurgus Cup (5th
- 4th
century B.C.). Such cup was
made from so-called “gold-ruby glass” contained gold nanoparticles (5-60 nm) causing the
color change based on the way of illumination. The glass appeared green in reflected light
and red when light was transmitted from inside.
The thigh connections between nanotechnology and physical chemistry can be found
through such great names as Albert Einstein with his Brownian motion theory and/or
Nobel prized Jean-Baptiste Perrin.
Nowadays, nanoparticles can be found everywhere from computers through house
facades coatings to clothing and cosmetics. They have their place in medicine as well as in
agriculture. Their benefits are indisputable including the advantages provided for
improvement of conventional, well-established methods and techniques. In the future,
however, the excessive use of nanomaterials may become problematic. Therefore,
powerful procedures have to be implemented, not only for the detailed characterization of
the properties of the produced nanoparticles but also for their sensitive detection in the
environment.
There is always more than one point of view. Therefore, this thesis is trying to look
at the symbiosis of nanomaterials and capillary electrophoresis from two perspectives –
analysis and characterization of nanomaterials and their exploitation for improved
performance of a well-known method.
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1.1 Structure of the thesis
The thesis is presented as a collection of selected peer-reviewed scientific papers
published between years 2011 and 2017. Since all discussed publications are attached, in
following text I am discussing only main achievements and general conclusions. The
included articles are referred to as “article X”
1.2 Personal commentary
During my PhD studies I was introduced to the field of electromigration techniques,
especially capillary electrophoresis. I realized that this family of methods is extremely
powerful when used properly (as every method). Its flexibility allows for its application for
analysis of analytes ranging from ions and small molecules through smaller or bigger
biomolecules including large proteins and nucleic acids to cellular compartments, viruses,
whole cells (both prokaryotic and eukaryotic), and even nano- and microparticles.
However, I have realized that even though the method is so powerful, there are still
some instrumental improvements, which can be done to enable even broader flexibility and
applicability. These improvements include combination of detection modalities as well as
development of alternative light sources for fluorescence detection. Some of these
shortcomings were addressed in publications included in my PhD thesis [1-4] and even
though my current work is based on these publications and I still draw information and
knowledge from what I have learnt during the my PhD studies, I have realized that present
trend of nanotechnologies, nanomaterials, and nanomedicine together with abilities of
capillary electrophoresis may create an efficient combination benefiting both sides.
First, capillary electrophoresis may provide excellent way of nanoparticle
characterization, synthesis quality control, and batch-to-batch preparation check-up
including polydispersity monitoring and interaction determination. Second, nanomaterials
not only due to their large surface area, but also due to the optical and electronic properties
may benefit to capillary electrophoresis by improving/enhancing the separation resolution
as well as detection sensitivity either as stationary/pseudo-stationary phases or labels
enabling fluorescent, chemiluminescent or electrochemical detection. Finally, the in-
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capillary environment is even suitable for low-volume nanoparticle preparation with online
characterization for green-chemistry applications.
My postdoctoral work at both Dublin City University as well as at Mendel
University in Brno was focused on development and application of capillary
electrophoretic method. Through this work, I was able to build a scientific group
(Laboratory of Bioanalysis and Imaging) at the Department of Chemistry and
Biochemistry of Mendel University in Brno currently accommodating 2 postdoctoral
researchers, 2 PhD students, 1 Master and 7 Bachelor students.
1.3 Author’s contribution
Currently, under my ORCID (0000-0002-6771-1304) combining my maiden name
(Ryvolova) and married name (Vaculovicova), one can find 98 hits at Web of Science
database including 60 journal articles, 14 reviews, 21 conference proceedings and 1
editorial material. From these I have chosen 7 experimental articles and 4 reviews that I
believe present pieces in the puzzle aiming at mapping the benefits of utilization of
capillary electrophoresis for investigation in the field of nanotechnologies or
nanomedicine.
The following summary is giving an overview of my contribution to the selected
works with special attention to amount of performed experiments, supervision of students,
definition of research direction, and my contribution to manuscript preparation.
1) Stanisavljevic M, Krizkova S, Vaculovicova M, Kizek R, Adam V. Quantum dotsfluorescence
resonance energy transfer-based nano-sensors and their application. Biosens
Bioelectron 2015;74:562-574.
Experimental work/Literature analysis 30%
Manuscript preparation 30%
Supervision 100%
Research direction 90%
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2) Dostalova S, Cerna T, Hynek D, Koudelkova Z, Vaculovic T, Kopel P, Hrabeta J, Heger
Z, Vaculovicova M, Eckschlager T, Stiborova M, Adam V. Site-directed conjugation of
antibodies to apoferritin nanocarrier for targeted drug delivery to prostate cancer cells.
ACS Appl Mater Interfaces 2016;8(23):14430-14441.
Experimental work/Literature analysis 30%
Manuscript preparation 30%
Supervision 50%
Research direction 70%
3) Dostalova S, Vasickova K, Hynek D, Krizkova S, Richtera L, Vaculovicova M,
Eckschlager T, Stiborova M, Heger Z, Adam V. Apoferritin as an ubiquitous nanocarrier
with excellent shelf life. Int J Nanomed 2017;12:2265-2278.
Experimental work/Literature analysis 30%
Manuscript preparation 30%
Supervision 30%
Research direction 30%
4) Konecna R, Nguyen HV, Stanisavljevic M, Blazkova I, Krizkova S, Vaculovicova M,
Stiborova M, Eckschlager T, Zitka O, Adam V, Kizek R. Doxorubicin Encapsulation
Investigated by Capillary Electrophoresis with Laser-Induced Fluorescence Detection.
Chromatographia 2014 Nov;77(21-22):1469-1476.
Experimental work/Literature analysis 50%
Manuscript preparation 80%
Supervision 80%
Research direction 70%
5) Janu L, Stanisavljevic M, Krizkova S, Sobrova P, Vaculovicova M, Kizek R, Adam V.
Electrophoretic study of peptide-mediated quantum dot-human immunoglobulin
bioconjugation. Electrophoresis 2013;34(18):2725-2732.
Experimental work/Literature analysis 30%
Manuscript preparation 90%
Supervision 50%
Research direction 60%
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6) Ryvolova M, Chomoucka J, Janu L, Drbohlavova J, Adam V, Hubalek J, Kizek R.
Biotin-modified glutathione as a functionalized coating for bioconjugation of CdTe-based
quantum dots. Electrophoresis 2011 Jun;32(13):1619-1622.
Experimental work/Literature analysis 60%
Manuscript preparation 80%
Supervision 50%
Research direction 50%
7) Stanisavljevic M, Chomoucka J, Dostalova S, Krizkova S, Vaculovicova M, Adam V,
Kizek R. Interactions between CdTe quantum dots and DNA revealed by capillary
electrophoresis with laser-induced fluorescence detection. Electrophoresis
2014;35(18):2587-2592.
Experimental work/Literature analysis 60%
Manuscript preparation 40%
Supervision 80%
Research direction 90%
8) Adam V, Vaculovicova M. Nanomaterials for sample pretreatment prior to capillary
electrophoretic analysis. Analyst 2017;142(6):849-857.
Experimental work/Literature analysis 100%
Manuscript preparation 90%
Supervision 80%
Research direction 90%
9) Adam V, Vaculovicova M. Capillary electrophoresis and nanomaterials - Part I:
Capillary electrophoresis of nanomaterials. Electrophoresis 2017 Oct;38(19):2389-2404.
Experimental work/Literature analysis 100%
Manuscript preparation 90%
Supervision 80%
Research direction 90%
10) Adam V, Vaculovicova M. CE and nanomaterials – Part II: Nanomaterials in CE.
Electrophoresis 2017 Oct;38(19):2405-2430.
Experimental work/Literature analysis 100%
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Manuscript preparation 90%
Supervision 80%
Research direction 90%
11) Nejdl L, Zitka J, Mravec F, Milosavljevic V, Zitka O, Kopel P, Adam V,
Vaculovicova M. Real-time monitoring of the UV-induced formation of quantum dots on
a milliliter, microliter, and nanoliter scale. Microchim Acta 2017;184(5):1489-1497.
Experimental work/Literature analysis 10%
Manuscript preparation 80%
Supervision 80%
Research direction 50%
List of abbreviations:
ACE – affinity capillary electrophoresis
CE – capillary electrophoresis
EDC – N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
Fab – antigen-binding region of antibody
Fc – region of antibody ensuring the communication with the other components of the
immune system
FRET – Fӧrster resonance energy transfer
QDs – quantum dots
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2 Nanotechnologies, nanomaterials and nanomedicine
2.1 Nanotechnologies
The term “Nanotechnology” was used in 1974 by Taniguchi and since that time it is used
for the scientific field where sizes from 0.1 to 100 nm play a crucial role. In the nano
range, gravity presents less an issue, however the strength of materials is more important
and also quantum size effect matters. The unique optical, electronic, thermal features and
chemical properties of nanomaterials as well as the ability to be chemically modified are
coming from their small dimensions and high ratios of surface to volume. Therefore,
nanomaterials found their applications in numerous scientific areas including physics and
engineering, however lately also in natural sciences including chemistry, biology and
medicine. Although nanomaterials are affecting various scientific fields, they are
approached differently. From the chemistry point of view, this field has historically been
related with colloids, micelles and/or polymers - typically, very large molecules, or
molecular aggregates. More recently, structures such as fullerenes, nanofibers, and
semiconductor quantum dots have been included in the group of particularly interesting
classes of nanostructures. In physics and electrical engineering, nanoscience is usually
related to quantum behavior and electrons and photons. Biology and biochemistry are also
interested in nanostructures as cell compartments; number of interesting biological
structures such as DNA, subcellular organelles and/or viruses can be labeled as
nanostructures [5-7].
2.2 Nanomaterials
“Nanomaterials” is a general name covering a large collection of compounds. Commonly
accepted definition is that a nanomaterial is “any material that has an average particle size
of between 1 and 100 nanometers.” Meanwhile, the definition given by The European
Commission states that 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.”[8]
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Firm following of such definitions permits the use of the term nanomaterials also to a
wide range biomolecules such as proteins or nucleic acids. For instance, the albumin
molecule is approximately 7 nm in size [9] and such protein fits the definition of
nanomaterial. However, it is not typically comprised into this group.
Simultaneously, some materials bigger than 100 nm, might also be included into the
nanomaterial group due their properties significantly different from properties of bulk
materials. For all these reasons, the definition given in the work by Buzea et al. may be
more appropriate [10]. The authors state that majority of the important properties of nano
begin to be apparent already in structures smaller than 1 μm (however some exceptions do
exist).
In general, nanomaterials cover materials of different natures and enormously varied
properties. Based on their specific properties, the materials as metallic and metal oxide
nanoparticles, semiconductor nanocrystals (quantum dots [11]), carbon nanomaterials
(nanodiamonds, fullerenes, graphene, carbon nanotubes) [12, 13], and polymeric
nanomaterials (e.g., chitosan, latex, polystyrene, dendrimers) can be distinguished [14, 15].
Based on shapes, dots, wires, tubes, ribbons, nanosheet, etc. may be identified. Among key
properties belong optical/fluorescent (e.g., quantum dots), electronic (e.g., fullerenes),
magnetic (e.g., metallic nanomaterials), and biological (e.g., liposomes).
2.2.1 Metal Nanoparticles
Undoubtedly, the biggest and most variable group of nanomaterials is the set of metal
nanoparticles. Metal- and metal-oxide nanoparticles are applicable as catalysts, sensors,
(opto)electronic materials, and for environmental remediation.
Nobel metals such as gold and silver are the most often used for generating
nanoparticles. Gold nanoparticles have been known about for 2500 years. The generation
of spherical gold nanoparticles is usually done via the citrate reduction method reported by
Turkevitch in 1951 [16]. The size distribution of the gold nanoparticles is controllable by
temperature, gold to citrate ratio, and the order in which reagents are added. Among other
methods belong the seeding technique [17], the two-phase reaction method [18], and an
approach employing inverse micelles [19] and dendrimers [20].
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Silver-based nanomaterials are attracting attention due to its characteristic properties,
such as chemical stability, catalytic activity, and good conductivity. Silver compounds
including nanoparticles are used also due to their antimicrobial properties. Chemical
reduction is the most commonly employed procedure for the synthesis of stable, colloidal
suspension either in water or organic solvents. Commonly used reductants include
borohydride, citrate, ascorbate, and elemental hydrogen [21].
Other noble metals such as platinum [22, 23], palladium [24-26], rhodium [27, 28],
and/or osmium [29-31] have also been described.
2.2.2 Semiconductor Nanocrystals
Semiconductor nanoparticles exhibit namely optical properties strongly associated with the
size and shape. These properties differ considerably from the bulk semiconductor material.
The shapes vary from nanorods, nanowires, and nanotubes to the currently very popular
quantum dots (QDs).
QDs have gained enormous popularity in the past two decades [32, 33]. Arising from
the quantum confinement of electrons and holes within the nanostructure, the fluorescence
of QDs is incomparable with organic fluorophores. In comparison with organic fluorescent
dyes and fluorescent proteins, QDs have molar extinction coefficients that are 10–50 folds
greater than those of conventional dyes, making them brighter and therefore applicable for
in vivo conditions. The long lifetime of 10 – 40 ns enhances the option of absorption at
lower wavelengths. Further, notable advantage is the high quantum yield from 40% to 90%
and the fact they are resistant to the influence of the ambient light (photobleaching) and/or
chemical degradation
Besides, QDs emission wavelengths are size-tunable over almost whole range of spectra
[34] and the emission wavelength can even reach the near-infrared region (650 nm to
950 nm). Large Stokes shift of QDs (300 – 400 nm) and ability of multiplex detection
enable imaging and/or monitoring several molecular targets simultaneously as well as
elimination of background autofluorescence, which limits in vivo fluorescence imaging
modalities. Since their introduction into biological imaging in 1998, enormous interest on
the aqueous synthesis [35, 36], characterization [37-40], and bioconjugation of QDs [35,
41, 42] was attracted.
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In article 1 of this thesis, a summary of utilization of quantum dots as sensors in Fӧrster
resonance energy transfer (FRET) applications is given.
FRET is a process during which the donor is excited by the external light source and the
emitted fluorescence is transferred to the acceptor leading to the emission of the light with
higher wavelength (emission of the acceptor). In the review, also systems using the
quenching mechanisms are included. In such case the acceptor returns after excitation to its
ground state via non-radiative decay pathways. In general, FRET is applicable for a
number of purposes including investigation of structural changes caused by alterations in
the molecular environment (e.g. temperature, pH, etc.) and/or exploration of molecular
interactions (i.e. aggregation or digestion).
2.2.3 Carbon nanomaterials
Especially due to its low toxicity for living organisms, nowadays carbon and carbon-based
materials are extensively investigated. Graphite attractively lies in the fact that it consists
of a flat single layer of carbon atoms organized into a two-dimensional (2D) honeycomb
structure - graphene. It can be wrapped up into 0D fullerenes, rolled into 1D nanotubes, or
stacked into 3D graphite. [43]
Fullerenes are arranged as a closed network of fused hexagons and pentagons. The
smallest stable and therefore most abundant fullerene is the buckminsterfullerene C60. [44]
Fullerenes display antiviral and antioxidant properties and because of the hollow cavity
inside the molecule, they serve as gene and drug carriers [45]. The lipophilic properties are
helpful for interactions with many enzymes or enable the intercalation into biological
membranes. Therefore, antibacterial activity of several derivatives may be observed [46].
Carbon nanotubes are well-ordered, hollow fiber-like nanomaterials consisting of tubes
of sp2
-hybridized carbon atoms [47]. The distortion of graphene into a cylinder noticeably
complicates the orbital overlapping, thus leading to carbon atoms wound around in a
helical mode. Moreover, a number of morphologies can occur among carbon nanotubes.
These are known as “hollow tube,” “bamboo” and “herringbone” [48].
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2.3 Nanomedicine
During the past years, substantial efforts have been driven towards the development of
effective therapeutic substances. However, current anti-tumor therapy possesses limited
safety and efficacy. Common conventional anticancer drugs display a narrow therapeutic
window because of the random distribution in the body. Non-specific distribution causes
cytotoxicity to healthy cells causing severe side effects to the patients. The non-specific
toxicity of anti-tumor drugs also restricts the applicable dose and thus lowers the
therapeutic value.
Nanoparticle-based drug carriers are colloidal systems acting as drug vehicles in the
form of nanospheres or nanocapsules [49]. Nanoparticle carriers belong most often to the
group of iron oxides, gold, biodegradable polymers, dendrimers, liposomes, viral capsids
[50-52] and/or proteins [53]. The formulation of the drug encapsulated in the nanocarrier
provides increased biocompatibility, which increases the applicability in clinical practice.
These types of drugs already used in the clinical practice include liposomal doxorubicin
and albumin-conjugate of paclitaxel [54].
Among the key properties of perfect nanocarrier is its size. The largest nanocarriers
are liposomes with their diameter of 80-200 nm [55], polymer-based particles (40-100 nm)
[56] or micelles (20-60 nm) [57] and the smallest ones are dendrimers (smaller than
10 nm) [58]. The size of nanocarrier should be uniform and enable the surface
modification with targeting molecules [59]. The use of suitable nanocarriers can
significantly decrease the undesired side effects, improve biocompatibility, specificity,
stability and water solubility [60].
The size of the nanocarrier should be low enough be able to enter the cells, but high
enough to avoid early removal from the body by renal clearance [61]. Furthermore, the
production should be simple and easy, capacity of encapsulation should be high and drug
release mechanism should be reliable to prevent undesired drug release but guarantee the
delivery into the target cells [62].
Even though, nanocarriers based on inorganic compounds/particles are easier for
preparation, they often cause an immune response of the organism or are even toxic to
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healthy cells. On the other hand, biomolecule-based nanocarriers can be found in human
body and therefore are more suitable [63]. Among such carriers belongs protein called
ferritin, which ensures the storage and transfer of iron ions [64]. After iron removal,
hollow cavity - apoferritin - is created [65]. Such cavity is ideal for drug transport mainly
due to the fact that the protein disassembles its structure in low pH environment. Such
environment can be found inside the cancer cells. Therefore, the effect of the carried drug
on the healthy cells is not as dramatic as on the diseased ones. Moreover, the encapsulation
of drugs in apoferritin does not require any modification of either drug or carrier molecules
because it employs an apoferritin behavior in surrounding environment [66]. The cell entry
is done via specific receptors, found on most body cells. However, this natural ability of
cell targeting and ferritin incorporation can be more enhanced by targeting moieties (e.g.
antibodies [67].
In article 2 of this thesis, targeted drug delivery system consisting of protein nanocarrier
(apoferritin), gold nanoparticles and antibodies was developed and characterized it
according to its long-term stability (article 3). The scheme of the developed targeted
nanocarrier is shown in Figure 1.
The apoferritin from horse spleen was used as a carrier of doxorubicin, which is an
effective anti-tumor drug with severe cardiotoxicity limiting its therapeutic dose.
Therefore, the protection of healthy tissues is highly required. The drug was encapsulated
into the protein cage, which was surface-modified by gold nanoparticles providing the
affinity to thiol groups of cysteine in the sequence of a heptapeptide (HWRGWVC). This
peptide was derived from the protein G, which exhibits a high affinity to the Fc fragment
of antibodies, which leads to the site-directed orientation of the antibody on the surface of
the apoferritin surface.
The conjugation of prostate specific membrane antigen antibodies and apoferritin was
used to target the prostate cancer cells (LNCaP) and HUVEC cells were used as a nontargeted
control. The encapsulation of doxorubicin in apoferritin with subsequent
modification by antibodies did not lead to lowering of doxorubicin toxicity for target
prostate cancer cells. On the other hand, nonmalignant cells were protected against the
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toxic effect of free doxorubicin. Moreover, the presented nanocarrier showed excellent
hemocompatibility.
Subsequently, detail study of the long-term stability of the developed nanocarrier
under various storage conditions was performed (article 3). The nanocarrier was prepared
in two solvents (water and phosphate buffer), and stored for 12 weeks at -20 °C, 4 °C,
20 °C, and 37 °C in dark and at 4 °C and 20 °C under ambient light. The parameters such
as optical properties; the amount of prematurely released drug molecules; size, shape, ζpotential,
and the ability to internalize into cancer cells and deliver the drug to nuclei were
tested. It was found out that the optimal storage conditions were 4 °C in dark and in water.
A very good stability for over 12 weeks was observed.
Figure 1: Scheme of the targeted apoferritin-based nanocarrier for doxorubicin transport
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3 Capillary electrophoresis in nanomedicine
Since the transfer of electrophoresis into the narrow capillaries by Jorgenson and Lukacs in
1981 [68], the technique has rapidly developed into a versatile analytical tool. Classical
capillary electrophoretic (CE) separation takes place in a fused silica capillary with internal
diameter of 20-100 μm, where the voltage of up to ±30 kV is applied. It is separating
molecules based on their mobilities in the electric field. Its main advantages include high
separation efficiency, short time of analysis and low consumption of chemicals.
Besides monitoring of properties of analytes such as size, charge, and/or surface
modification, etc., CE is able to monitor the interactions between molecules. An entire
field has appeared known as “affinity capillary electrophoresis” (ACE) [69, 70]. Although
many interactions are being investigated and employed, the term ACE appears to be more
or less reserved for stronger interactions with specific stoichiometries.
Nanomaterial surfaces can be easily modified and functionalized by many molecules
that may enter into interactions with molecules of interest. In this case, CE can work in
several ways: 1) monitoring the direct interaction between the nanomaterial and analyte
(the signal is provided by both the analyte and nanoparticle), 2) monitoring the interaction
between two analytes mediated by the nanomaterial (the signal is provided by the analytes
themselves, not by the nanomaterial), and 3) monitoring the interaction between two
analytes reported by a change in the signal of the nanoparticle.
3.1 Cargo encapsulation
The aim of the article 4 of this thesis was to study the encapsulation of doxorubicin into
the cavity of apoferritin nanotransporter and to investigate the behavior of doxorubicin in
CE using electrolytes with different pH values with purpose of exploration of release of the
drug from the apoferritin cage by changing pH. It can be summarized that decreasing the
pH of doxorubicin-carrying apoferritin to 3 caused the presence of a single peak with
migration time matching to the doxorubicin at the same conditions. Therefore, the release
of the drug was confirmed. Further, the properties of doxorubicin using capillary
electrophoresis with laser-induced fluorescence detection were investigated. The intrinsic
20
fluorescence of the drug enabled to monitor the behavior of doxorubicin in the presence of
several compounds quenching the fluorescence.
3.2 Conjugation to targeting ligands
To improve the targeted drug delivery, the conjugation of the nanotransporter with a
selective targeting ligand is recommended. For such conjugation, antibodies, targeting
peptides (e.g. RGD peptide, etc.), or small molecules (e.g. folic acid, etc.), nanocarriers can
be used to specifically target the diseased cells [20–23]. The coupling can be done by
covalent bond, physical or hydrophobic adsorption. Commonly, cross-linking through an
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC)/Nhydroxysuccinimide
reaction is used. In the EDC coupling, there is an unwanted option
that the antigen binding sites of antibodies are blocked by the nonselective formation of
amide bonds close to a Fab (antigen-binding fragment) area of the antibody. For this
reason, alternative ways are searched.
Therefore the article 5 of this thesis presents capillary electrophoretic investigation of
the nanostructure conjugation with antibody with the aim of sterically-specific orientation
to ensure the activity of the antigen-binding fragment of the antibody remains unchanged.
This approach was employed in the designing of the above mentioned drug nanocarrier
(doxorubicin-carrying apoferritin).
In the article, a new strategy of the coupling of CdTe QDs with human
immunoglobulin using a specially designed heptapeptide was presented. The heptapeptide
with a sequence of HWRGWVC was prepared and characterized by mass spectrometry,
liquid chromatography, and capillary electrophoresis. Subsequently, the peptide was used
as a stabilizing compound for QDs. The coupling QDs capped via this peptide with
immunoglobulin was studied by capillary electrophoresis and magnetic immunoextraction
coupled with differential pulse voltammetry. Finally, the prepared conjugates were used
for fluorescent detection using immobilized goat antihuman immunoglobulin antibody.
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4 Monitoring of the interaction between nanoparticles and
biomolecules
4.1 Affinity interaction
Besides the affinity interaction between Fc fragment of antibody and HWRGWVC peptide,
other affinity-exhibiting pairs are applicable. The (strept)avidin–biotin interaction is
considered as the strongest non-covalent interaction with dissociation constant Kd = 4×10-
14
M. (Strept)avidin-biotin complex is rapidly formed and it is resistant to many extreme
conditions (extreme pH values, temperature and even to denaturing agent). Due to strong
and reliable affinity (strept)avidin-biotin interaction is often used in diagnostics
application. In most of the assays when interaction is applied, streptavidin is coupled to
solid phase, such as QDs, magnetic particles, microtitration wellplate and surfaces while
biotin is conjugated to the molecule of the interest (e.g. protein, etc.).
However, in the article 6, different approach was taken. Biotin-conjugated
glutathione was prepared and used as a coating for CdTe QDs. Such coating connected the
ability of the biotin to bind avidin, streptavidin and/or neutravidin with the fluorescent
properties of the QDs creating specific, high-affinity fluorescent label. The results obtained
by the capillary electrophoretic analysis of the prepared probe showed that biotinylated
glutathione is suitable coating for the elegant synthesis of thiol capped QDs. Obtained QDs
were of good properties for fluorimetric detection and moreover, it was demonstrated that
capillary electrophoresis is an efficient method for separation of the glutathione and
biotinylated glutathione excess from the quantum dots stabilized with biotinylated
glutathione. Moreover, the functionality was verified by interaction with avidin.
4.2 Non-specific interaction
In some cases, not only the covalent labeling or affinity-based conjugation is required, but
also non-specific interaction based on structural properties of the analyte and probe may be
of an interest. However, it should be highlighted that not only intentional coupling of
nanoparticles with biomolecules may occur, but due to the constantly increasing use of
nanomaterials, the undesired interactions may happen. Therefore, both of these
possibilities (intentional and unintentional interaction) should be taken into an account.
22
One of the possible targets is the genetic information of the cell. Possible interaction
between DNA and nanomaterials is probably ensured by electrostatic binding in major
groove of double stranded DNA (dsDNA) and incorporation between base pairs.
The aim of work presented in article 7 was aqueous synthesis of CdTe QDs capped
with glutathione of the specific 2 nm size for monitoring of the interaction based on QDs
size fitting into the major groove of the DNA double helix. Characterization of the
nanoparticles has confirmed desired size of 2 nm. The interaction between QDs and DNA
have been studied, through time and different concentration interaction with double
stranded genomic chicken DNA (dsDNA), ssDNA and 500 base pair long DNA fragment.
The comparison of the interaction between ssDNA and dsDNA has confirmed that dsDNA
is needed for complex creation because peak complex was not observed in the case of
ssDNA interaction with QDs. Interaction with 500 base pairs long DNA fragment has
shown the same tendency of creating complex as with genomic DNA. The presence of the
QDs in the structure of DNA was observed with gel electrophoresis after ethidium bromide
staining. Observed interaction relies on possible similarity between size of quantum dots
and major groove of the DNA (aprox. 2.1 nm).
5 CE for nanomaterials and nanomaterials for CE
5.1 Sample pretreatment by nanomaterials
CE can be coupled with many detection techniques. Each of them has its own advantages
as well as disadvantages in terms of sensitivity, selectivity, and/or versatility. On-capillary
as well as off-capillary detection modes are available. On-capillary detection is a
nondestructive strategy minimizing the band broadening and enabling the employment of
several detectors simultaneously (either consecutively or at the same detection point,
however the off-capillary methods may provide additional information such as molecular
mass. Photometric (or absorbance) detection is outstanding because of its versatility and
due to this reason it is the most commonly used technique. However, the internal diameter
of the capillary (generally tens of μm) defines the light path length. Therefore, relatively
high analyte concentrations are necessary. Otherwise, extended path length flow cells
(bubble cell, Z-cell), or preconcentration techniques are required to reach satisfactory
23
results. Moreover, several cleaning steps are usually needed in case of biological matrices
significantly interfering with electrophoretic analysis. Sample pretreatment and
preconcentration can be done either electrophoretically or chromatographically.
Electrophoretic strategies are based on un-matching electrophoretic mobilities of the
components or on their behavior in presence of (pseudo)stationary phase. On the contrary,
chromatographic methods rely on compound sorption on a solid-phase material. These
techniques take advantage from loading of multiple capillary volumes of sample
subsequently eluted in a minute volume of solvent.
For extraction, isolation or preconcentration purposes, nanomaterials are offering
appreciated high surface-to-volume ratios. As an example may serve the comparison of
surface area of carbon microparticles with 60 μm in diameter of (0.01 mm2
) and the
surface area of carbon nanoparticles with 60 nm in diameter (11.3 mm2
). Besides the
increase in the surface area, the reactivity increases approximately 1000×. Not only the
surface area, but also the chemical affinity may be beneficial.
For example, gold nanoparticles deliver outstanding isolation power because of the
high affinity for thiols. Similarly, magnetic separation is a method using magnetism for the
effective separation mediated by paramagnetic and superparamagnetic particles. This
technique relies on the option of surface modification of magnetic nanoparticles to
facilitate immunoextraction. Such particles may be modified by either antibodies for
targeted capture of the analyte of interest or by oligonucleotide chains having sequences
complementary to the desired nucleic acid. Magnetic particles can be immobilized using a
magnetic field while interfering compounds are removed from the solution [71]. The above
mentioned preconcentration techniques can be combined with CE in an off-line, at-line,
on-line, or in-line mode.
The summary of the work combining the nanomaterial-based sample pretreatment
(preconcentration) followed by CE analysis is the goal of the article 8 of this thesis.
5.2 Characterization of nanoparticles by CE
Despite of all the advances in nanomaterial synthesis, problem in terms of batch-to-batch
repeatability still persists. Moreover, sometimes, tools for characterization of nanomaterial
24
composition and properties are missing. Even in the same lot, the polydispersity of the
particles and the inconsistency of the properties may present problem for satisfactory
application. Therefore, it is not surprising that various methods of synthesis and
characterization of the nanomaterials have been developed. From these methods, capillary
electrophoresis has its unreplaceable position due to many advantages discussed. CE is the
easy to use and low cost technique for studying nanoparticles parameters, such as their
size, surface chemistry, and interaction abilities. Based on the literature search, it can be
concluded that electromigration techniques represent a family of effective methods for
nanomaterial characterization, evaluation, and investigation. In the future, manufacturing
of portable or even hand-held CE-based instruments for in situ characterization of
produced nanomaterials, as well as incorporating of CE into some industrial devices for
high-throughput production of nanomaterials as quality control may emerge this field even
further.
The overview of application of CE for separation and characterization of nanoparticles is
given in article 9 of this thesis.
5.3 Separation improvement
Even though, the development of miniaturized devices using microfluidic chips presented a
great boom at the end of last century, nowadays, an increasing number of researchers
perform CE separations in short capillaries (units of centimeters) instead of microfluidic
chips [72, 73]. In such capillaries, fast and efficient separations take place without more or
less difficult chip preparation requiring expensive facilities (e.g. clean rooms and
lithography). In comparison to microchip-based rapid CE, short capillary-based high-speed
CE take advantage of simple structure, easy fabrication, and low costs.
The shortcoming, however, is in lowered resolution linked with short separation
length. This obstacle can be overcome either by injection of very low sample volumes
(picoliters) or by extra selectivity coming from an added (pseudo)stationary phase of
various nature (e.g. micelles, nanoparticles, nanostructures, etc.). Such solution
significantly eliminates the adsorption on the capillary wall [74-77]. Nanomaterials have
been proven to be effective (pseudo)stationary phase due to their beneficial properties,
such as large surface/volume ratio and simple functionalization. Among often used
25
nanomaterials belong carbon nanotubes [78]. But, also other structures including
nanoparticles [79, 80], nanofibres [81] and/or nanorods [82, 83] have been used. On the
other hand, interaction of analyte with immobilized nanostructures such as monoliths,
nanopillars, bound nanoparticles and/or other nanomaterials is also applicable. All of these
types have already been employed in coupling with CE. Immobilized nanomaterials, either
deposited on capillary wall as a thin layer coating or filled into the capillary, are frequently
utilized as stationary phases for capillary electrochromatographic mode of separation.
Equally, (pseudo)stationary phases enable a broad range of functionalities providing a
number of interactions [84].
5.4 Enhancement of detection sensitivity
Laser-induced fluorescence detection is the most sensitive optical detection modality
connected with microcolumn separations. Picomollar detection limits [85] and good
detection selectivity enables analysis of samples in rather complex matrices. Concurrently,
this selectivity could be perceived as a limitation due to the fact that majority of analytes
does not fluoresce and derivatization by some fluorescent label is desirable.
Such photoluminescent labels may be not only organic fluorophores or fluorescent
proteins but also QDs [86-89]. However, besides photoluminescence detection,
chemiluminescence and electrochemiluminescence detection are also benefiting from
properties of nanomaterials [90]. Undesired background signals are eliminated which leads
to improved sensitivity. Furthermore, the instrumentation is simplified by absence of
certain optical components such as excitation sources or optical filters. Metal
nanomaterials (such as gold, silver, platinum, semiconductors, and magnetic) are in
chemiluminescence and electrochemiluminescence detection applicable as catalysts,
fluorophores, or energy acceptors [91].
Potentiometric, amperometric and conductometric are three of the most commonly
used types of electrochemical detection in CE. Compared to the optical detection modes is
that the electrochemical detection is mostly performed by off-column, end-capillary, and
therefore, in destructive arrangement. The main roles of nanoparticles cover biomolecule
immobilization, catalysis of reactions, improvement of electrode-analyte electron transfer,
analyte labelling, and even use as a reactant [92].
26
It is highly unlikely that nanomaterials will wholly substitute such well-established
approaches as organic dyes for fluorescent labeling. However, nanomaterials offer new
options for a broad range of applications. The electrochemical detection particularly
benefits from use of nanomaterials that enable increasingly sensitive detection.
The abilities of nanomaterials to improve the performance of CE analysis in terms of both
separation resolution as well as detection sensitivity are summarized in the article 10 of
this thesis (Figure 2)
Figure 2: Summary of application of nanomaterials for improvement of CE performance
27
6 In-capillary quantum dot synthesis
6.1 Organometallic synthesis of quantum dots
Organometallic synthesis is still the most popular method of quantum dot preparation. It
was introduced by Murray et al. in 1993 and involves very high temperature, toxic
precursors, organic solvents and surfactants. However resulting quantum dots are highly
monodisperse, size-tunable and surface coated. Usually the precursors are loaded into the
flask with organic solvents trioctylphosphine (TOP) and its oxide (TOPO). The reaction is
performed under an inert atmosphere and at 230-260 °C for nanocrystals growth. Final
hydrophobic quantum dots have to be subsequently modified for water-solubility and
biocompatibility. Nevertheless problems of the reproducibility, lack of the control and the
overall costs of the procedure are its major disadvantages.
6.2 Aqueous synthesis of quantum dots
The second most often used synthesis method for direct producing stable and
biocompatible aqueous solution of QDs is aqueous synthesis. Reactions are performed in
three-necked flask with reflux condenser. Heavy metal precursors are easily dissolved in
water; while chalcogens precursors can be bought as commercial solid powder. Metal salts
dissolution in water occurs in the presence of capping agents (usually thiols, e.g. 2mercaptoethanol,
2-thioglycerol, thioglycolic acid). The thiols control the QDs synthesis
kinetics, passivate surface, provide stability, solubility and surface functionality of the
QDs. This method is considered more environment-friendly and less expensive than
organometallic, easy with high reproducibility, but comparing to the organometallic
procedure lower quantum yields and higher polydisperity is observed. Disadvantages of the
aqueous synthesis including a long reaction time (hours and/or days) have been overcome
by employing microwave irradiation. High-quality quantum dots with enhanced quantum
yield are produced. Generally, CdTe, CdSe, CdS, Zn1-xCdxS and ZnSe QDs are
synthesized by microwave irradiation.
28
6.3 The other ways of synthesis
Above mentioned methods are very effective in production of highly fluorescent QDs but
usage of toxic chemicals limits their application in clinical practice. Therefore, more ecofriendly
paths of QDs synthesis have been investigated using the principles of „green
chemistry‟ such as use of biocompatible and non-toxic solvents, precursors, and stabilizers
such as bovine serum albumin as a capping agent.
Also, biosynthesis is new area of synthesis method and involves biological
organisms and their metabolic pathways in preparation of QDs of desired composition,
size, shape and functionality. Microbial synthesis is done either intracellular or
extracellular and each of these QDs has their specific characteristics and is naturally
capped with the proteins from the system with good stability and compatibility for any
biological application. However, challenges such as improving size and shape control,
obtaining larger amount of QDs and detail explanation of the synthesis mechanisms have
to overcome.
6.4 UV irradiation in-capillary synthesis of quantum dots
In the article 11 included in this thesis, a detail investigation of the inexpensive, lowtemperature,
and rapid preparation of aqueous QDs by UV illumination is reported. The
influence of UV irradiation (at 254 nm and 250 nm) and temperature on the solutions of
precursors were described. Optimal results were achieved with a solution of precursors
composed by cadmium, selenium and mercaptosuccinic acid (quantum yield of 13.5%).
Furthermore, the synthesis and observation of the formation of QDs in quantities from submg
to sub-ng was carried out. The smallest concentration being 258 pg in volume of 4 nL.
The growth of QDs was observed in real time by photometry, fluorimetry and dynamic
light scattering. Among biggest advantages of the presented method belong the simplicity,
controllability, and low costs. The presented procedure can be integrated with miniaturized
analytical systems or other instrumentation.
29
7 Conclusion
Undoubtedly, nanomaterials are exceptionally valuable tool not only improving highly the
selectivity and effectivity of analyte isolation methods, increasing the ability of separation
techniques to distinguish closely-structured molecules but also and improving greatly the
abilities of detectors. Among numerous of crucial features of devices used for clinical
purposes belong simplicity of application and robustness. Although, the great benefits of
capillary electrophoresis are indisputable, robustness and repeatability of analyses belong
to the weak points. Therefore, use of CE in clinical practice is limited to DNA sequencer.
Therefore, employment of nanomaterials in CE might open new insight due to lower
detection limits on one side and enhance the separation efficacy on the other side.
Nevertheless, this is at the beginning and waiting for exploration.
Nanomaterials such as liposomes and dendrimes are able to advance the separation part of
the CE analysis and QDs can significantly improve the detection part. Nevertheless, some
types of nanomaterials such as carbon nanotubes or metal nanoparticles can improve both.
Simultaneously, CE is an effective technique for characterization of nanomaterials,
evaluation and/or investigation.
The relationship of CE and nanotechnology is advantageous not only for analytical
chemists and material scientists, but also for biochemists and molecular biologists, because
it leads to the advance of new, more effective, and more sensitive approaches.
The combination with the ease of miniaturization is offering opportunities for portable and
point-of-care applicable devices suitable for personalized diagnostics. Moreover, the
improved separation power of electrophoretic analysis promises effective analyses of
complex biological samples.
30
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9 Articles
36
Article 1
Stanisavljevic M, Krizkova S, Vaculovicova M, Kizek R, Adam V. Quantum dotsfluorescence
resonance energy transfer-based nano-sensors and their application. Biosens
Bioelectron 2015;74:562-574.
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Article 2
Dostalova S, Cerna T, Hynek D, Koudelkova Z, Vaculovic T, Kopel P, Hrabeta J, Heger Z,
Vaculovicova M, Eckschlager T, Stiborova M, Adam V. Site-directed conjugation of
antibodies to apoferritin nanocarrier for targeted drug delivery to prostate cancer cells.
ACS Appl Mater Interfaces 2016;8(23):14430-14441.
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Article 3
Dostalova S, Vasickova K, Hynek D, Krizkova S, Richtera L, Vaculovicova M,
Eckschlager T, Stiborova M, Heger Z, Adam V. Apoferritin as an ubiquitous nanocarrier
with excellent shelf life. Int J Nanomed 2017;12:2265-2278.
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Article 4
Konecna R, Nguyen HV, Stanisavljevic M, Blazkova I, Krizkova S, Vaculovicova M,
Stiborova M, Eckschlager T, Zitka O, Adam V, Kizek R. Doxorubicin Encapsulation
Investigated by Capillary Electrophoresis with Laser-Induced Fluorescence Detection.
Chromatographia 2014 Nov;77(21-22):1469-1476.
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Article 5
Janu L, Stanisavljevic M, Krizkova S, Sobrova P, Vaculovicova M, Kizek R, Adam V.
Electrophoretic study of peptide-mediated quantum dot-human immunoglobulin
bioconjugation. Electrophoresis 2013;34(18):2725-2732.
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Article 6
Ryvolova M, Chomoucka J, Janu L, Drbohlavova J, Adam V, Hubalek J, Kizek R. Biotinmodified
glutathione as a functionalized coating for bioconjugation of CdTe-based
quantum dots. Electrophoresis 2011 Jun;32(13):1619-1622.
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Article 7
Stanisavljevic M, Chomoucka J, Dostalova S, Krizkova S, Vaculovicova M, Adam V,
Kizek R. Interactions between CdTe quantum dots and DNA revealed by capillary
electrophoresis with laser-induced fluorescence detection. Electrophoresis
2014;35(18):2587-2592.
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Article 8
Adam V, Vaculovicova M. Nanomaterials for sample pretreatment prior to capillary
electrophoretic analysis. Analyst 2017;142(6):849-857.
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Article 9
Adam V, Vaculovicova M. Capillary electrophoresis and nanomaterials - Part I: Capillary
electrophoresis of nanomaterials. Electrophoresis 2017 Oct;38(19):2389-2404.
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Article 10
Adam V, Vaculovicova M. CE and nanomaterials – Part II: Nanomaterials in CE.
Electrophoresis 2017 Oct;38(19):2405-2430.
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Article 11
Nejdl L, Zitka J, Mravec F, Milosavljevic V, Zitka O, Kopel P, Adam V, Vaculovicova M.
Real-time monitoring of the UV-induced formation of quantum dots on a milliliter,
microliter, and nanoliter scale. Microchim Acta 2017;184(5):1489-1497.
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