· XV. Workshop of Physical Chemists and Electrochemists 4 Brno 2015 THE ORGANIZATION HOSTING THE...
Transcript of · XV. Workshop of Physical Chemists and Electrochemists 4 Brno 2015 THE ORGANIZATION HOSTING THE...
Brno, 26th and 27th of May, 2015
Book of abstracts
XV. Workshop of Physical
Chemists and
Electrochemists
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Brno
2015
XV. Workshop of Physical Chemists and Electrochemists
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XV. Workshop of Physical Chemists and
Electrochemists
Book of abstracts 26th and 27th of May, 2015
Masaryk University
Brno, 2015
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THE ORGANIZATION HOSTING THE CONFERENCE
Faculty of Science, Masaryk University in Brno
Department of Chemistry
Kotlářská 2
611 37 Brno
http://www.sci.muni.cz
THE ORGANIZATIONAL SECURITY OF THE
CONFERENCE
Libuše Trnková
(Department of Chemistry, Faculty of Science, Masaryk University)
The publication did not undergo the language control. All contributions are publicated in the
form, in which they were delivered by the authors. Authors are also fully responsible for the
material and technical accuracy of these contributions.
© 2015 Masaryk University
ISBN 978-80-210-7857-4
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The Workshop of Physical Chemists and Electrochemists was
supported by research organizations and projects:
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The sponsors of the Workshop of Physical Chemists and Electrochemists:
The organizers thank a lot to all this year‘s sponsors for the support, which enabled to
organize this traditional conference: Metrohm Czech Republic s. r. o., Eppendorf Czech &
Slovakia s r.o., Sigma - Aldrich spol. s r.o., Pragolab s.r.o, MANEKO spol. s r. o., HACH –
LANGE s r. o., CHROMSPEC spol. s r. o. and Czech Chemical Society, subdivision Brno.
Main sponsor
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Úvodem...
Další rok od 14. Pracovního setkání je za námi a my se opět setkáváme v hojném
počtu na 15. ročníku Workshop of Physical Chemists and Electrochemists. Masarykova
univerzita Vás srdečně vítá na konferenci, která se koná u příležitosti životního jubilea
profesora Viktora Brabce, význačného českého biofyzika, biofyzikálního chemika a u
příležitosti 125tého výročí narození profesora Jaroslava Heyrovského, nositele Nobelovy ceny
za fyzikální chemii. Čekají nás dva dny plné přednášek, prezentací studentů v soutěžní sekci
mladých, plakátových sdělení, majících snahu podpořit výzkum v oblasti fyzikální chemie,
biofyzikální chemie a elektrochemie. Jménem organizátorů bych Vám chtěla všem popřát
úspěšnou konferenci, která Vás bude inspirovat k dalšímu vědeckému bádání společně s
navázáním nové spolupráce. Jednoduše, nechť se Vám konference líbí a je pro Vás přínosem.
Libuše Trnková
Motto:
„Učenec v laboratoři není jen odborník, je to i dítě, které hledí na vědu, jako na pohádku.
Vidí v ní krásu.“
Marie Curieová - Sklodowská
An introductory word...
A year from the 14th Working Meeting of Physical Chemists and Electrochemists is
behind us and we are meeting again in the 15th Workshop of Physical Chemists and
Electrochemists in a large number of participants. Masaryk University kindly invites you to
the conference, in this year, held on the occasion of the 70th birth anniversary of professor
Viktor Brabec, the prominent Czech biophysicist and biophysical chemist, and on the
occasion of the 125th birth anniversary of professor Jaroslav Heyrovsky, the Nobel Prize for
Physical Chemistry winner. There are two days ahead filled by lectures, presentations of
students in competition section of young researches, poster presentations tending to support
our research in the field of Physical, Biophysical chemistry and Electrochemistry. On behalf
of the organizers I would like to wish you a successful conference that will inspire you in
further scientific research supported by new co-operations. Simply, let me wish you a nice
conference, which could be of benefit to you.
Libuše Trnková
Motto:
„A scientist in his laboratory is not only a technician: he is also the child placed before
natural phenomena, which impress him a fairy taile.“
Marie Curie - Sklodowska
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Table of contents
Transition metal-based constructs as anticancer drug candidates. Recent advances and insights
12
Novel approaches to electrode modification by nanostructured metals 14
Spectroelectrochemistry and electrofluorimetry of biologically relevant fluorescent dyes 17
Novel electrochemical DNA biosensors as useful tools for investigation and detection of DNA
damage 18
Scanning electrochemical microscopy 22
Plasma treatment of carbon nanomaterial screen-printed working electrodes of
electrochemical sensors 25
Fluorescence polarization assay to quantify binding of selected fluorescent ligands to
haloalkane dehalogenases with modified tunnels 29
Simple electrochemical transducing system with optical readout for point-of-care applications
32
A biosensing application of pencil graphite electrode modified by copper nanoparticles for
adenine detection 34
Atomic force microscopy for characterization of biomolecules, affinity complexes and cells 38
Switching of electrochemical properties of proteins upon glycation 40
Transfer of monovalent ions in quadruplex DNA systems 44
Detection of glucose using gel-templated gold nanostructured electrodes 47
Electrochemical corrosion of steel as a source of Fe2+
catalyst of Fenton reaction 51
Development of fluorescent substrates of haloalkane dehalogenases for mechanistic
enzymology 55
Analysis of biodegradable nanofibrous layers 57
Electrochemical Li insertion into TiO2 polymorphs: Study of mechanism and structural
changes 61
Voltammetric behaviour of herbicide linuron on boron-doped diamond electrode 64
Designing nucleobases for nucleic acid quadruplexes 68
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The comparison of chemical and magnetic channelrhodopsin-2 transfection efficiency 71
Contributions of cytochromes p450 to detoxification of a human carcinogen aristolochic acid
I in human and rat livers 75
Optical and spectroelectrochemical study of interaction between meso-
tetrakis(4-sulphonatophenyl)porphyrin derivatives and cyclodextrins in aqueous solution 79
Vacuolar-ATPase-mediated intracellular sequestration of ellipticine contributes to drug
resistance in neuroblastoma cells 82
Role of zinc ions in advanced prostate cancer model 86
The influence of vertex potential and multiple scan voltammetry on the formation of 8-
oxoguanine 87
Voltammetric detection of DNA damage caused by 2-aminofluorene and its metabolite 2-
acetylaminofluorene 91
Multi-walled carbon nanotubes and their double-step functionalization with etoposide and
antisense phosphorothioate oligodeoxynucleotides 95
Evaluation of ANTI-PAIIL immunoglobulin efficacy by monitoring of luminescent
Pseudomonas Aeruginosa 99
Benzo[a]pyrene is oxidized by rat hepatic microsomes both in the presence of NADPH and
NADH 103
Fabrication of nanoporous alumina membranes for electrochemical sensors 107
On the anodizing behaviour of aluminium in citric acid electrolytes 111
A central role for phytochelatin in plant and animals: A review 115
Study of characterization mammalian metallothioneins by MALDI-TOF/TOF and
electrochemical method 118
Core/shell quantum dots as fluorescent labels of biomolecules 122
Benzo[a]pyrene, ellipticine and 1-phenylazo-2-naphthol induce expression of cytochrome b5
in rats 126
Lead pencil graphite as electrode material: Structural and electrochemical properties 130
Cheap and quick production of micro amalgam electrodes for determination of soil
contaminated with heavy metal ions (Cd(II) and Pb(II)) 133
Synthesis of conjugated aromatic systems and their properties 137
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Diamond coated quartz crystal microbalance sensor for detection of protein adsorption 142
Electrochemical fabrication of thin nanoporous titania surfaces 146
Voltammetry of guanosine and guanosine monophosphate on a pencil graphite electrode 150
Modification of CQDs monitored by Brdicka reaction 154
Prion protein and its interactions with metals and metallothionein 3 158
Microfluidic chip with amperometric detection for monosaccharides determination 161
Voltammetric determination of bicarbonate 165
NADPH- and NADH-dependent oxidation of DNA adduct formation by benzo[a]pyrene
catalyzed with human cytochrome p450 1A1 169
Modification of carbon electrode with graphene oxide sheets 173
Get to know Metrohm 177
UV-VIS spectrophotometry of microliter sample volume by means of NanoDrop instruments
(Thermo Scientific) 178
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TRANSITION METAL-BASED CONSTRUCTS AS ANTICANCER
DRUG CANDIDATES. RECENT ADVANCES AND INSIGHTS
Viktor BRABEC
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 612 65 Brno,
Czech Republic
Abstract
The application of bioinorganic and organometallic chemistry to medicine is a field which has
yet to realize its potential and is lagging well behind classical inorganic and organic
chemistry. The overall objective is to establish new principles for the design of metal-based
compounds with exquisite therapeutic properties and to deliver truly novel methods for
activation and targeting of therapeutic metal complexes. Ideally these complexes will have a
high degree of selectivity for the desired target and distinct mechanisms of action which are
unlike those of any existing therapeutic agents. To achieve this, a wide range of innovative
methodology is used since progress in this field is currently greatly retarded by the lack of
suitable methods to study the structures and interactions of metal-based compounds under
biologically-relevant conditions.
The development of metal-based chemotherapeutics has been stimulated by the clinical
success of cis-diamminedichloridoplatinum(II) (cisplatin) and its analogues and by the
clinical trials of other platinum complexes with activity against resistant tumors and reduced
toxicity including orally available platinum drugs. The widespread clinical applications of
cisplatin and its simple analogues have inspired the synthesis and investigation of numerous
transition-metal based compounds as potential drug candidates. In particular, there is much
interest in expanding the tumors that can be treated, limiting side effects, and targeting the
cancer cell population. Broadening the spectrum of antitumor drugs depends on understanding
existing agents with a view toward developing new modes of attack. In other words, a better
understanding of the processes underlying biological effects of existing drugs would guide the
choice of new compounds for more effective therapies. It is therefore of great interest to
understand details of molecular and biochemical mechanisms underlying the biological
efficacy of the transition metal-based compounds. There is a large body of experimental
evidence that the success of platinum and other transition-metal based complexes in killing
tumor cells results from their ability to form on DNA various types of covalent adducts so that
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the research of DNA interactions of metal-based antitumor drugs has predominated. This
contribution will present current knowledge on DNA modifications by selected new transition
metal-based complexes of biological significance, their recognition by specific proteins and
repair. It will also provide a strong support for the view that metallodrugs which bind to DNA
in a fundamentally different manner to that of “classical” cisplatin will have altered
pharmacological properties. It will be also demonstrated that this concept has already led to
the synthesis of several new unconventional transition metal-based antitumor compounds that
violate the original structure-activity relationships. In addition, this critical contribution will
consider besides the existing platinum drugs also specific (inter-related) areas of research of
selected metallodrugs which are challenging, have a ground-breaking nature, and potentially
high impact. These areas are: Polynuclear platinum complexes, photoactivated therapeutic
metal complexes and supramolecular, substitution-inert metallohelices. The rapid evolution of
the field is being informed by post-genomic knowledge and approaches, and further dramatic
step-change breakthroughs can be expected as a result; harnessing this knowledge and
responding to and taking advantage of this new environment requires integration of chemistry
and biology research.
ACKNOWLEDGEMENT
The work has been supported by the Czech Science Foundation (Grant 14-21053S) and the
Ministry of Education, Youth and Sports of the Czech Republic (Grant LH13096).
REFERENCES
Brabec V, Kasparkova J: Drug Resist. Updates, 8 (2005), 131-146
Brabec V. Novakova O.: Drug Resist. Updates, 9 (2006), 111-122
Hannon MJ: Pure Appl. Chem., 79 (2007), 2243–2261
Bednarski PJ, Mackay FS, Sadler P et al.: Anti-Cancer Agents Med. Chem., 7 (2007), 75-93
Lovejoy KS, Lippard SJ: Dalton Trans., (2009), 10651 - 10659
Howson SE, Bolhuis A, Brabec V, et al.: Nature Chemistry, 4 (2012), 31-36
Brabec V, Howson SE, Kaner RA et al.: Chem. Sci., 4 (2013), 4407-4416
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NOVEL APPROACHES TO ELECTRODE MODIFICATION BY
NANOSTRUCTURED METALS
Jan HRBÁČ1*
1Department of Chemistry, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic. E-mail:
Abstract
The electrochemical/electrophoretic deposition method and DC electric (spark) discharge
were proposed as techniques allowing to modify conductive substrates with metal
nanostructures. Both techniques can be regarded as facile, green and low-cost.
1. INTRODUCTION
After initial research focus in development of reproducible preparation methods of well
separated and stable nanoparticles of inorganic and organic materials, the current research is
oriented at preparation of nanoparticles' organized assemblies, most often nanostructured
layers on the surface of a suitable solid substrate. Nanostructured metallic layers on
conductive substrates belong to the above mentioned research trend, targeted into the
development of novel, highly sensitive electrochemical and surface enhanced Raman
scattering (SERS) sensors. Two novel methods of the preparation of nanostructured metal
films on conducting substrates, which can be regarded as facile, green and low-cost were
developed in our laboratories.
The electrochemical/electrophoretic deposition method is characterised by using a sacrificial
anode undergoing progressive electrochemical dissolution in ultrapure water as a source of
metal material. The cations formed by electrolysis form insoluble dispersions of
corresponding oxides/hydroxides, which fill the interelectrode space by the electrophoretic
movement, diffusion and convection induced by density and temperature gradients during
electrolysis and are in equilibrium with corresponding metal cations. On the cathode
substrate, the cations are reduced to form nanostructured metal films. The deposition of silver
onto silicon substrates was studied in detail [1], where discrete nanosized (5-25 nm) silver
particles attached to the surface were obtained at lower applied voltages (5-20 V) while
deposition at 30 V resulted in silver nanowires with mean diameter of 75 nm and lengths up to
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15 µm.. The latter deposits exhibited high electrocatalytic activity towards hydrogen peroxide
reduction and applicability as SERS [1,2] substrates was also demonstrated. Using the
proposed approach, a broad range of metals can be deposited, namely Ag, Au, Cu, Bi, Zn, and
Sn. As cathodes, carbon fibers were used due to their attractive sensing properties and easy
visualisation by scanning electron microscopy. Coating of carbon fibers with copper yielded
irregular nanowire-like deposit allowing for efficient amperometric detection of carbohydrates
both in batch and flow arrangements [3].
The next developed approach utilizes the DC electric (spark) discharge between the two
electrodes (i.e. between a screen printed or carbon fiber substrate electrode and a source metal
electrode) in ambient or inert atmospheres. Heat, introduced due to the flow of electricity
leads to the formation of air plasma and vaporized nanoparticles by each electrode material
(i.e. carbon and metal), the percentage of which depends on the polarity, with the material of
the electrode connected to the negative pole of the power supply being in excess. When the
sparking process is performed in ambient atmosphere, the oxidation of metal in vapour may
occur. Of metals which can be deposited by the sparking process, bismuth was deposited onto
screen printed electrodes. With bismuth connected to positive pole and screen printed
electrode to negative pole of the power supply rated at 1200 V with internal resistance of
1 MΩ, extremely small bismuth nanoparticles 5 nm dispersed in carbon were formed on the
screen printed electrode substrate. Exceptionally high performances of these electrodes in
heavy metal anodic stripping analysis [4] and adsorptive stripping of organic analyte -
riboflavin [5], both in non-deoxygenated solutions.
2. ACKNOWLEDGEMENT
The work has been supported by Grant Agency of the Czech Republic, projects P206-12-0796
and 15-05198S. The presenter wishes to thank all colleagues involved in the experimental
work.
3. REFERENCES
[1] Halouzka V, Jakubec P, Kvitek L, et all.: J. Electrochem. Soc., 160 (4) (2013), B54-B59.
[2] Halouzka V, Trnkova L, Hrbac J, et all.: Substrát pro povrchem zesílenou Ramanovu spektroskopii. CZ
Patent: (2014) 304500-B6.
[3] Riman D, Bartosova Z, Halouzka V et all.: RSC Adv. 5 ( 2015), 31245-31249.
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[4] Riman D, Jirovsky D, Hrbac J, et all.: Electrochem Comm. 50 (2015), 20-23.
[5] Riman D, Avgeropoulos A, Hrbac J, et all.: Electrochim. Acta 165 (2015), 410-415.
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SPECTROELECTROCHEMISTRY AND ELECTROFLUORIMETRY
OF BIOLOGICALLY RELEVANT FLUORESCENT DYES
Teresa OBERSCHMID1, Tomáš SLANINA
1,2*
1 Institute of Organic Chemistry, Faculty of Chemistry and Pharmacy, Universität Regensburg,
Universitätsstraße 31, 93053 Regensburg, Germany
2 Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 267/2, 611 37 Brno, Czech
Republic
ABSTRACT
Fluorescent dyes became highly important in last few decades.1,2
They are broadly used for
cell staining, flow cytometry, fluorescence microscopy, super-resolution imaging and bio-
sensing for in vivo analytics. The absorption and emission properties are often modulated by
change of pH,3 quenching of excited state
4 as well as the change of redox state by electron
transfer (eT).5 The knowledge of identity and fate of paramagnetic reactive intermediates
generated by redox reaction is essential for understanding and design of new systems. A novel
technique complementary to spectroelectrochemistry, electrofluorimetry, is introduced. It
enables to measure absorption and emission properties of intermediates generated by a redox
reaction on the working electrode. Investigation of rhodamine 6G radical anion is used as a
model system relevant for bio-applications.
REFERENCES
[1] Gonçalves, M. S. T.: Chem. Rev., 109 (2009), 1, 190-212
[2] Dsouza, R. N., et al.: Chem. Rev., 111 (2011), 12, 7941-7980
[3] Han, J., and Burgess, K.: Chem. Rev., 110 (2010), 5, 2709-2728
[4] Lavis, L. D., and Raines, R. T.: ACS Chemical Biology, 3 (2008), 3, 142-155
[5] Auchinvole, C. A. R., et al.: ACS Nano, 6 (2012), 1, 888-896
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NOVEL ELECTROCHEMICAL DNA BIOSENSORS AS USEFUL
TOOLS FOR INVESTIGATION AND DETECTION OF DNA DAMAGE
Vlastimil VYSKOČIL1*
1 Charles University in Prague, Faculty of Science, University Research Centre UNCE "Supramolecular
Chemistry", Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry,
Hlavova 2030/8, 128 43 Prague 2, Czech Republic
Abstract
Damaging interactions of various xenobiotic compounds with DNA are among the most
important aspects of biological studies in clinical analysis, drug discovery, and
pharmaceutical development processes. In recent years, a growing interest in electrochemical
investigation of such supramolecular interactions has arisen. Simple electrochemical DNA-
based biosensors, recently developed in our UNESCO Laboratory of Environmental
Electrochemistry and applied to detect DNA damage, will be introduced in this contribution.
1. INTRODUCTION
A huge diversity of problems currently solved by modern bioanalytical chemistry requires a
great variety of approaches, methods, and materials used for finding optimal solutions.
Although the advantages and possibilities of current spectrometric and separation methods are
fascinating, it can certainly be declared that modern electrochemical methods may represent a
competitive alternative, especially if they use novel electrode materials and progressive
approaches [1]. In our UNESCO Laboratory of Environmental Electrochemistry, we have
recently been intensely interested in novel types of electrochemical DNA biosensors based on
various carbon transducers for detection of DNA damage [2-6]. The possibilities and limitations
of these newly developed biosensors, their advantages and drawbacks, their practical
applications as well as their contribution to contemporary bioanalytical chemistry will be
presented to provide a detailed look at new approaches in the development of modern
electrochemical biosensing systems.
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2. NOVEL ELECTROCHEMICAL DNA BIOSENSORS
Fabrication of biosensors
DNA biosensors are integrated receptor–transducer devices that use DNA (isolated, e.g., from
calf thymus [2,3] or salmon sperm [4,5]) as a biomolecular recognition element to measure
specific binding processes with DNA. Compared with other transducers, electrochemical ones
received particular interest due to rapid detection and great sensitivity. Among the electrochemical
transducers, carbon-based electrodes (e.g., a glassy carbon electrode [5], a screen-printed carbon
electrode [2,4], or a microcrystalline natural graphite–polystyrene composite film electrode
[3]) exhibit several unique properties. Their wide electrochemical potential window in the
positive direction allows sensitive electrochemical detection of DNA damage by monitoring
the appearance of oxidation peaks of DNA bases [1].
Adsorption is the simplest method to immobilize DNA on the carbon electrode surface. It does
not require reagents or special modifications in the DNA structure. The surface of the carbon
electrode is usually pretreated by applying a positive potential (ca. 1.5 to 1.8 V vs. Ag|AgCl)
for a certain time [2]. This pretreatment of the carbon surface increases its roughness and
hydrophilicity. Afterwards, the electrochemical adsorption of DNA is realized using a stirred
solution at a potential of 0.5 V (vs. Ag|AgCl) for a preset time [5] that depends on DNA
concentration. This potential enhances the stability of the immobilized DNA through the
electrostatic attraction between the positively charged carbon surface and the negatively
charged hydrophilic sugar-phosphate backbone. Another way to immobilize DNA by
adsorption is realized by evaporation of a small volume of DNA solution on the electrode
surface [2,3]. Nanostructured interfaces between the bare electrode and DNA, formed by various
nanomaterials (e.g., carbon nanotubes [4]), represent another approach to the enhancement of
the biosensor response due to inherent electroactivity, effective electrode surface area, etc.
Detection techniques
Voltammetric detection modes (especially cyclic voltammetry, differential pulse
voltammetry, and square-wave voltammetry) are the most frequently used. Together with them,
electrochemical impedance spectroscopy becomes to be popular at DNA-based biosensors [4].
According to electrochemically active species, which responses are evaluated at DNA damage
detection, the experimental techniques can be classified as follows [1]: (i) label-free and often
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reagentless techniques which represent the work with no additional chemical reagents (redox
indicators, mediators, enzyme substrates, etc.) needed to generate measured response and (ii)
techniques which employ redox indicators either non-covalently bound to DNA (groove
binders, intercalators, anionic or cationic species interacting with DNA electrostatically) or
presents in solution phase (e.g., [Fe(CN)6]4–/3–
anions). Combination of these principles allows
obtaining more complex information on DNA changes and damaging supramolecular
interactions [4].
The first group of techniques utilizes surface activity or redox activity of DNA itself [1].
Electrochemical oxidation on carbon electrodes showed that all bases (guanine, adenine,
cytosine, and thymine) can be oxidized. As the oxidation is irreversible, measurements cannot
be performed repeatedly. Initial increase in the anodic guanine moiety response after a short-time
incubation of the biosensor in damaging agents can indicate opening of the original double-
stranded DNA structure, while decrease in this response is an evidence for the deep DNA
degradation [4]. Some products of DNA damage exhibit characteristic electrochemical signals
(e.g., anodic peaks of 8-oxo-7,8-dihydroguanine [2] and 2,8-dihydroxyadenine moieties) which
can be evaluated with better sensitivity than the change in responses of original DNA bases.
The second group of techniques employs electroactive compounds added to the measured
system and interacting with DNA non-covalently as its indicators (anionic or cationic
indicators, intercalators, or groove binders). A decrease in the indicator response indicates
double-strand break formation and helix destruction. The redox indicators may be also used as
diffusionally free species present in solution phase. For instance, [Fe(CN)6]4–/3–
anions indicate
the presence of DNA layer on the electrode surface on the basis of electrostatic repulsion
between the indicator anion and the negatively charged DNA backbone [4]. Moreover, the
investigated xenobiotic compound itself can serve as a redox indicator [4,5].
3. CONCLUSIONS
Electrochemical DNA-based biosensors nowadays represent very effective and at the same
time simple, fast, inexpensive, miniaturizable, and mass-producible bioanalytical devices for
evaluation and classification of genotoxic effects of individual chemical (e.g., carcinogens, dyes,
pesticides, or various industrial chemicals) or physical agents (e.g., UV radiation). Moreover,
evaluation of DNA protection capacity of various natural and synthetic chemical substances
(antioxidants) is also possible using detection of DNA damage caused by pro-oxidants.
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4. ACKNOWLEDGEMENT
Financial support of this contribution by the Grant Agency of the Czech Republic (Project
GP13-23337P) is gratefully acknowledged.
5. REFERENCES
[1] Palecek E, Bartosik M: Chemical Reviews, 112 (2012), 6, 3427-3481
[2] Vyskocil V, Labuda J, Barek J: Analytical and Bioanalytical Chemistry, 397 (2010), 1, 233-241
[3] Vyskocil V, Barek J: Procedia Chemistry, 6 (2012), 52-59
[4] Hlavata L, Benikova K, Vyskocil V, Labuda J: Electrochimica Acta, 71 (2012), 134-139
[5] Hajkova A, Barek J, Vyskocil V: Electroanalysis, 27 (2015), 1, 101-110
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SCANNING ELECTROCHEMICAL MICROSCOPY
David HYNEK1,2
and Rene KIZEK1,2*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
Over the past few years, the field of nanomaterials has largely expanded due to the special
physical and chemical properties of micro- and nano-structured materials that strongly
contrast with those of bulky materials. Visualisation of these structures based on the currents
measurements is the aim task for scanning electrochemical microscopy (SECM). This
technique can effectively show the studied surface electrochemical properties. Different
strategies were reported to immobilize particles on the electrode surface depending on various
ways of functionalization. Electrochemical detection of the redox label generates a specific tip
current, whose intensity depends on the local surface concentration of the redox
macromolecule. Detected electrochemical signal created electrochemical images with specific
current levels.
1. INTRODUCTION
The first papers related to the scanning electrochemical microscopy (SECM) were published
in the nineties of the 20th
century [1]. This technique is based on the scanning of the studying
surface by ultramicroelectrode (UME) and electrochemical detection of surface options. Such
technique gives us the electrochemical picture of the surface. SECM employs an UME probe
(tip) to induce chemical changes and collect electrochemical information while approaching
or scanning the surface of interest (substrate). The substrate may also be biased and serve as
the second working electrode. Electrodes in micro or nano dimensions offer important
advantages for electroanalytical applications including greatly diminished ohmic potential
drop in solution and double-layer charging current, the ability to reach a steady state in
seconds or milliseconds, and a small size allowing make experiments in microscopic
dimensions.
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Due to the non-destructive measurement method can be successfully experiment repeated
several times. The SECM surface scanning is connect with one major risk of damage to the
scanning electrodes or the studied surface, due to distance between the tip and surface
interference.
2. PRINCIPLE OF SECM
An UME tip is attached to a 3D piezo positioner controlled by a computer, which is also used
for data acquisition. A bi-potentiostat controls the potentials of the tip and/or the substrate
versus the reference electrode and measures the tip and substrate currents in pA orders. The
most SECM measurements take place in feedback (FB) or generation/collection (GC) modes.
The feedback mode usually used the UME which serves as the working electrode in a three or
four-electrode system. The four electrode system is completed with the sample (the substrate)
which serves as a second working electrode. The electrodes are immersed in a solution
containing redox mediator. Specifically, one redox form of a quasi-reversible redox couple. In
the simplest case, the UME is only inserted in mediator solution, a potential is applied to the
tip, and diffusion-controlled conversion of the mediator occurs according to equation as
follows: R O + ne- (1)
and thus a steady-state faradaic current iT could be detected. This situation is necessary to
understand as the limiting case where the distance d of the tip from the substrate electrode is
infinite.
Generation/collection mode differs from feedback mode in one important thing, the presence
of the mediator is solution. This mode works in a solution that does not initially contain any
substance that can be converted at the UME at a potential ET. In the generation/collection
(G/C) mode, both tip and substrate can be used as work electrodes, one work electrode
generates some species which are collected at the other electrode. The G/C mode is more
sensitive because the background signal is very weak.
The spatial resolution of SECM is mainly affected by the probe size. i.e., a smaller probe
offers a higher spatial resolution. Besides the probe size, the tip–substrate distance is another
crucial parameter of SECM procedure. The tip must be positioned and maintained in close
proximity to the substrate to obtain a high-resolution image.
XV. Workshop of Physical Chemists and Electrochemists
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3. APPLICATIONS
Application of SECM is very wide and allows study various structures and processes in micro
and submicrometer-sized systems. The target of detection could be electron, ion, and
molecule transfers, and other reactions at solid-liquid, liquid–liquid, and liquid-air interfaces.
In recent years the study of nanoparticles and its application rapidly increases [2]. The wide
range of SECM applications allows investigate a wide variety of processes, the corrosion of
metals, material characterization, membrane reactions, enzymatic reactions, photosynthesis,
DNA hybridization and metabolism in single living cells [3]. This technique allows the
imaging of an individual cell on the basis of not only its surface topography but also such
cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis
and membrane transport. The bio-applications of SECM are just one of the most developed
areas in the recent years and the usage of nanoparticles in this area is very widespread [2-4].
This direction has had the great influence for the increase of published papers related to the
SECM technique since the year 1995 (approximately) [1].
4. ACKNOWLEDGEMENT
The work has been supported by NanoBioTECell P102/11/1068.
5. REFERENCES
[1] Sun P, Laforge F O, Mirkin M V, Phys. Chem. Chem. Phys., 9 (2007), 802-823.
[2] de la Escosura-Muniz A, Ambrosi A, Merkoci A, Trac-Trends Anal. Chem., 27 (2008), 568-584.
[3] Edwards M A, Martin S, Whitworth A L, et al., Physiol. Meas., 27 (2006), R63-R108.
[4] Amemiya S, Guo J D, Xiong H, et al., Anal. Bioanal. Chem., 386 (2006), 458-471.
XV. Workshop of Physical Chemists and Electrochemists
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PLASMA TREATMENT OF CARBON NANOMATERIAL SCREEN-
PRINTED WORKING ELECTRODES OF ELECTROCHEMICAL
SENSORS
Ondřej JAŠEK1,2*
, Marek ELIÁŠ1,2
, Lenka ZAJÍČKOVÁ1,2
, Petra MAJZLÍKOVÁ3,4
, Jan
PRÁŠEK3,4
, Jaromír HUBÁLEK3,4
1 Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
2 Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
3 Central European Institute of Technology, Brno University of Technology, Technická 3058/10, 616 00 Brno,
Czech Republic
4 Centre of Sensors, Information and Communication Systems, Faculty of Electrical Engineering and
Communication, Technická 3058/10, 61600 Brno, Czech Republic
Abstract
Plasma treatement was used to modify screen-printed working electrode (WE) of an
electrochemical sensor. The WE material consist of various carbon nanomaterials including
carbon nanotubes and carbon nanosheets related nanostructures. The plasma treatment was
performed using radio frequency capacitive coupled discharge in Ar/O2 and
Ar/cyclopropylamine. The surface of the WEs was characterized by scanning electron
microscopy and their electrochemical behaviors were studied using potassium
ferro/ferricyanide. Best results were achieved using oxygen plasma where significant
improvement of current response was achieved.
1. INTRODUCTION
Wide range of structures, including carbon nanotubes[1], multi-layered nanosheets and
graphene[2], exhibit great potential for electrochemical sensing. Fabrication of carbon
nanomaterial electrochemical sensors is often performed using functionalized or modified
CNTs. The most common approach is an acid treatment that removes end-caps and leads to an
attachment of carboxylic groups (-COOH). Next to the chemical functionalization, a plasma
treatment is well known as the method of choice to functionalize and tailor material surfaces.
In our work plasma treatment of used for modification of electrochemical sensors screen-
printed electrodes.
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2. MATERIAL AND METHODS
Material
The MWCNTs were purchased from Nanocyl s.a. (Belgium) as NC7000. Carbon nanotubes
were also directly synthesized on WE of electrochemical sensors using microwave plasma
torch in Ar/H2/CH4 deposition mixture.
Potassium ferrocyanide K4[Fe(CN)6] and potassium ferricyanide K3[Fe(CN)6] were provided
by Sigma–Aldrich. N,N-dimethylformamide, potassium chloride KCl and hydrochloric acid
HCl were obtained fromPenta (Czech Republic). 3-Hydroxytyraminium chloride (dopamine –
DA) was purchased from Merck Schuchardt OHG (Germany). Thick-film pastes, ESL 9562-
G, ESL 243-S and ESL 5545-G, were purchased from ESL Electro-science (UK) whereas
DuPont 7102 carbon paste and DuPont 5874 silver/silver chloride paste were delivered byDu
Pont (UK) Ltd.
Plasma treatment
The modifications were carried out in a low-pressure RF (13.56MHz) capacitive coupled
(CCP) discharge. The reactor was a glass tube, 80mm in diameter and 185mm in length,
enclosed by aluminium ganges serving as RF and grounded electrodes. The treated material
was placed in the middle of the tube. A further description of the reactor can be found in the
previous paper [3].
Electrochemical measurement
Electrochemical measurements were performed with AUTOLAB PGSTAT 204
potentiostat/galvanostat controlled by Nova 1.10 software (Metrohm Autolab B.V.,
Netherlands). The electrodes were characterized by cyclic voltammetry using a three-
electrode voltammetric cell with standard Ag/AgCl reference electrode (type 6.0729.100,
Metrohm, Switzerland) and platinum auxiliary electrode (type 6.0343.000, Metrohm,
Switzerland) in the equimolar solution of 2.5mmol/L potassium ferro/ferricyanide in 0.1
mol/L KCl electrolyte solution (pH 5.8). The scan rate was 50 mV/s and potential range was
from -1.0V to +1.0V for K4[Fe(CN)6]/K3[Fe(CN)6].
XV. Workshop of Physical Chemists and Electrochemists
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3. RESULTS AND DISCUSSION
Prepared carbon nanomaterials samples were analyzed by scanning electron microscopy and
Raman spectroscopy. Working electrodes of electrochemical sensors were then fabricated
using these materials and plasma treated in (CCP) discharge.
A representative cyclic voltammogram of directly grown multi-walled carbon nanotubes
(MWCNT) sensor were recorded at 50 mV·s−1
with 2.5 mM [Fe(CN)6]4−
/3−
(1:1) solution in
0.1 M KCl . Two well-defined symmetric redox peaks separated by ΔEp = (Epa − Epc) = 83
mV were observed. The ratio of the anodic and cathodic peak currents reached unity (Ipa/Ipc =
1.01). The stability of the MWCNT electrode was studied for the [Fe(CN)6]4−
/3−
(1:1) solution
in 0.1 M KCl at 50 mV·s−1
using 10 cycles of CV. The results revealed that both, the
oxidative and reductive, peak currents of the studied redox couple remained practically
constant throughout all 10 potential cycles [4].
To compare directly grown WE and their performance, plasma treatment was carried out
using screen-printed and spray-coated electrodes. The most promising results were obtained
with the electrodes modified by oxygen plasma because the reversibility and the current
response were both improved significantly. The peak-to-peak separation improved from 879
to 116mV after the oxygen plasma treatment of the bare DuPont 7102 electrode. Spray-coated
MWCNTs had a positive effect on the performance of the screen-printed DuPont 7102 WE if
the sprayed MWCNTs layer was thicker. The modification by Ar/NH3 and
Ar/cyclopropylamine lead to only small improvement or negatively influenced electrode
performance [5].
4. CONCLUSION
Plasma treatment was used to modify carbon nanomaterial working electrode of an
electrochemical sensor. Best results were achieved using oxygen plasma where significant
improvement of current response was achieved.
5. ACKNOWLEDGEMENT
This work was supported by the project ‘CEITEC – Central European Institute of
Technology’ CZ.1.05/1.1.00/02.0068 and by the SIX project CZ.1.05/2.1.00/03.0072.
XV. Workshop of Physical Chemists and Electrochemists
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6. REFERENCES
[1] Iijima S.: Nature, 354 (1991), 56-58
[2] Novoselov K. S., Geim A. K., Morozov S., et.al.: Science, 306 (2004), 666–669
[3] Zajíčková L., Eliáš M., Buršíková V., et.al: Thin Solid Films, 538 (2013), 7-15
[4] Majzlíková P., Sedláček J., Prášek J., et.al.: Sensors, 15 (2015), 2644-2661
[5] Majzlíková P., Prášek J., Eliáš M., et. al.: Phys. Status Solidi A, 211(12) (2014), 2756–2764
XV. Workshop of Physical Chemists and Electrochemists
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FLUORESCENCE POLARIZATION ASSAY TO QUANTIFY BINDING
OF SELECTED FLUORESCENT LIGANDS TO HALOALKANE
DEHALOGENASES WITH MODIFIED TUNNELS
Shubhangi KAUSHIK1, Jiri DAMBORSKY
1, Radka CHALOUPKOVA
1*
1Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic
Compounds in the Environment RECETOX, Masaryk University, Brno, Czech Republic
Abstract
Fluorescence polarization (FP) is a method for rapid and non-destructive quantitative analysis
of the interaction of small fluorescent ligands with a larger biomolecules in a real time [1].
Here we describe FP assay for analysis of binding kinetics of selected HaloTag ligands into
haloalkane dehalogenase enzymes with modified tunnels. A set of biochemically
characterized variants of haloalkane dehalogenase DhaA [2-4] and LinB [5] with modified
access tunnels were studied in the present work to analyze their ability to accommodate
various fluorescence ligands by FP method.
1. INTRODUCTION
Haloalkane dehalogenases are enzymes that catalyze hydrolytic cleavage of the carbon-
halogen bonds in halogenated aliphatic hydrocarbons releasing a halide ion, a corresponding
alcohol and a proton as the reaction products. Active sites of these enzymes are deeply buried
inside the protein interior and connected with surrounding environment by access tunnels,
which play important role in functionality of the enzymes [6]. In order to assess how
introduced mutations in the access tunnels of haloalkane dehalogenase affect their ability to
accommodate small ligands, FP method was used to determine binding kinetics of selected
enzymes with various HaloTag ligands. All studied variants of haloalkane dehalogenase carry
the mutation in the catalytic histidine (His272Phe) resulting in formation of an ester bond
between the nucleophile and the substrate, which cannot be further hydrolyzed, providing the
covalent alkyl-enzyme intermediate [2].
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2. MATERIALS AND METHODS
Mutagenesis of catalytic histidine to phenylalanine at position 272 in studied haloalkane
dehalogenases was carried out using QuikChangeTM
Site-directed mutagenesis kit (Stratagene,
USA). The nucleotide sequences of the mutants were confirmed by DNA sequencing (GATC,
Germany). The genes encoding mutants of selected haloalkane dehalogenases were
transformed into expression host and protein expression was induced by addition of isopropyl
β-D-1-thiogalacto-pyranoside (IPTG) in culture media. The proteins were purified using
metallo-affinity chromatography and their proper folding was determined by circular
dichroism (CD) spectroscopy. FP analysis was conducted at room temperature using Infinite
F500 plate reader (Tecan, Switzerland) equipped with polarizers for excitation and emission.
The purified enzymes were reacted with selected HaloTag ligands in phosphate-buffered
saline containing 0.01% CHAPS detergent to minimize the non-specific interactions.
MALDI-TOF MS spectra were recorded for selected haloalkane dehalogenases on an
Ultraflextreme instrument (Bruker Daltonics, Germany) operated in the linear mode with
detection of positive ions to confirm formation of covalent complex between studied enzymes
and HaloTag ligands.
3. RESULTS AND DISCUSSION
The constructed histidine-substituted variants of haloalkane dehalogenases with modified
tunnels were successfully overexpressed in E.coli host cells and purified to homogeneity. CD
spectroscopy in far-UV region revealed that His272Phe substitution has no effect on overall
secondary structure of the enzymes. Obtained results from FP analysis indicated that the
method is sufficiently sensitive to monitor differences in binding kinetics of HaloTag ligands
to various haloalkane dehalogenases based on the nature of mutation present in their access
tunnels. The differences in binding kinetics were detected even when a single point mutation
was introduced into the access tunnel. Moreover, the binding kinetics was found to be
influenced by the type of fluorescent ligand, employed during the binding reaction. The
MALDI-TOF MS analysis confirmed successful binding of fluorescence ligands into
haloalkane dehalogenases with large tunnel opening and no formation of covalent complexes
between the ligands and haloalkane dehalogenases carrying the bulky substitutions introduced
into their tunnels.
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4. CONCLUSIONS
The present work used haloalkane dehalogenase as model proteins, but FP method is broadly
applicable for monitoring binding kinetics with other proteins where acyl enzyme
intermediate could be trapped. This method thus has a good potential for analysis of
accessibility of tunnels and active sites in enzymes forming the covalent intermediates.
5. ACKNOWLEDGEMENTS
This work was supported by the Grant of the Ministry of Education of the Czech Republic
(CZ.1.07/2.3.00/30.0037).
6. REFERENCES
[1] Perrin F: Journal de Physique et le Radium, 7 (1926), 390-401
[2] Los GV, et al.: ACS Chemical Biology 3 (2008), 373-382
[3] Pavlova M, et al.: Nature Chemical Biology 5 (2009), 727-733
[4] Liskova V, et al.: ChemCatChem 7 (2015), 648-659
[5] Chaloupkova R, et al.: Journal of Biological Chemistry 278 (2003), 52622-52628
[6] Prokop Z, et al.: Protein Engineering Handbook (2012), 421-464
XV. Workshop of Physical Chemists and Electrochemists
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SIMPLE ELECTROCHEMICAL TRANSDUCING SYSTEM WITH
OPTICAL READOUT FOR POINT-OF-CARE APPLICATIONS
Karel LACINA1*
, Zoltán SZABÓ2, Jaromír ŽÁK
1,3, Pavel FIALA
2, Petr SKLÁDAL
1
1 Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
2 Department of Theoretical and Experimental Electrical Engineering, Faculty of Electrical Engineering, Brno
University of Technology, Technicka 3058/10, 616 00 Brno, Czech Republic
3 Department of Microelectronics, Faculty of Electrical Engineering, Brno University of Technology,
Technicka 3058/10, 616 00 Brno, Czech Republic
A simple device for the transduction of an electrochemical signal to a visual readout suitable
for point of care diagnostics has been designed (Fig 1, left) [1,2]. The transducer consisting of
only a 4-electronic components circuit - two resistors, one transistor and one light emitting
diode (LED) - amplifies and optically indicates faradaic currents flowing through an
electrochemical cell (Fig. 2, right). Analytical performance of the device – sensitivity,
threshold level and limit of detection - could be simply modulated by careful adjustment of
the value of two resistors (R1 and R2) connected with the base electrode of the transistor. The
biosensing abilities of the proposed system were tested on the proof-of-concept model using
immobilised glucose oxidase. The negligible construction costs and high simplicity are the
principal benefits of the proposed platform. This approach is innovative in the
transduction/conversion of the signal to visual perception and also in the signal generation –
neither potentiostat nor galvanostat are used compared to the majority of electrochemical
measurements.
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Figure 1: Simplest transduction of the electrochemical event using a bipolar transistor amplifier
consisting of the electrochemical cell (EC), resistors (R1and R2), light emitting diode (LED) and
transistor (T). Eappl denotes applied potential – a power supply, e.g. 3 V lithium battery (left).
Measurement with the proposed transducer where T, R1 and R2 were BC547, 2.575 MΩ and 721 kΩ,
respectively. The concentrations of zones of H2O2 in the electrochemical cell are depicted in mM
(right).[1,2]
ACKNOWLEDGEMENT
This work was realised in CEITEC (CZ.1.05/1.1.00/02.0068) with the support from the
project "A new types of electronic circuits and sensors for specific applications" no. FEKT-S-
14-2300 and was financed by the National Sustainability Program under grant LO1401. For
the research, infrastructure of the SIX Center was used as well.
REFERENCES
[1] Lacina K, Skládal P, Sensors and Actuators B 210 (2015), 183-189
[2] Lacina K, Skládal P, Systém pro převod elektrochemického signálu na vizuální vjem, užitný vzor, PUV
(2014), 2014-30355
XV. Workshop of Physical Chemists and Electrochemists
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A BIOSENSING APPLICATION OF PENCIL GRAPHITE ELECTRODE
MODIFIED BY COPPER NANOPARTICLES FOR ADENINE
DETECTION
Vimal SHARMA 1, Marian MAREK
2, Jan CECHAL
3, and Libuse TRNKOVA
1,4 *
1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, CZ–625 00 Brno, Czech
Republic
2CEITEC and Department of Microelectronics, Brno University of Technology, Technicka 3058/10, 616 00
Brno,
3CEITEC and Institute of Physical Engineering, Brno University of Technology, Technicka 3058/10, 616 00
Brno,
4SIX Research Centre, Brno University of Technology, Technicka 3058/10, CZ–616 00 Brno, Czech Republic
Abstract
A pencil graphite electrode (PeGE) modified by copper nanoparticles (Cu NPs) was prepared
to determine adenine. The results indicated that modification of PeGE by Cu NPs exhibits an
enhancement in the oxidation peak current with a positive shift of the peak potential, in
contrast to that observed on original PeGEs at pH ranges from 3.0 to 8.0 investigated by
cyclic voltammetry. The simplicity and sensitivity of the modified electrode promises the
probability of the detection of electroactive biomolecules.
1. INTRODUCTION
Purine derivatives play an important role in the regulation of biological functions [1, 2] and
understanding their redox behavior is a primary task for electrochemical methods [3]. The
increase of sensitivity and selectivity in oxidation responses of adenine (Ade) was achieved
by a chemical modification of the pencil graphite electrode (PeGE). This electrode was
modified by copper nanoparticles (Cu NPs) which possess great potential application in the
field of photovoltaics, chemical sensing and biosensors. The metallic NPs used were
stabilized by the polypyrrolidone (PVP) shell. Cu NPs modified electrodes were also
characterized by UV-Vis, zetasizer, and X-ray photoelectron spectroscopy and scanning
electron microscopy, and a novel electroanalytical tool was employed to determine adenine on
a Cu NPs modified electrode.
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2. MATERIAL AND METHODS
Adenine, copper sulphate pentahydrate (CuSO4.5H2O), diethylene glycol ((HOCH2CH2)2O),
sodium borohydride (NaBH4), sodium hypophosphite (NaPO2H2), sodium hydroxide (NaOH),
methanol (CH3OH), acetic acid (CH3COOH), phosphoric acid (H3PO4), Mili Q water.
Synthesis of Cu NPs:
The synthesis of ultrafine copper nanoparticles was typically processed in organic solvent by
dissolving a certain amount of poly(N-vinyl pyrrolidone) (PVP, MW = 40,000), acting as the
capping molecule, in diethylene glycol (DEG) in a round bottom flask. Afterward, at room
temperature, copper(II) sulfate (1 ml of 0.1 M aqueous solution) was added under strong
magnetic stirring followed by adjusting the pH of the solution up to 11 with dropwise addition
of 0.1 M NaOH in DEG solution. Under continuing stirring, 0.1 M NaBH4 DEG solution was
quickly added into the flask. In the first few minutes, the deep blue solution gradually became
colorless, and then it turned burgundy, suggestive of the formation of a copper colloid.
Linear sweep voltammetry (LSV) & cyclic voltammetry (CV)
All measurements were performed in a potentiostat Autolab PGSTAT30 (Metrohm, Czech
Republic) using a three-electrode system with an auxiliary platinum electrode, a reference
Ag/AgCl/KCl (3M) electrode, and a pencil graphite electrode (PeGE, diameter 0.5 mm,
surface area 16 mm2) from Tombow (Japan) as the working electrode. The
measurements were performed in 0.1 M phosphate acetate buffer.
3. RESULTS AND DISCUSSION
Copper nanoparticles (Cu NPs) were synthesized by the chemical reduction method. Their
size was determined by Zetasizer (MALVERN, Nano-ZS) and corresponds to 23-25 nm.
Further, Cu NPs were characterized by SEM, XPS, and UV/Vis. The SEM images verified the
adsorption of Cu NPs on PeGE surfaces (Figure 1).
XV. Workshop of Physical Chemists and Electrochemists
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Figure 1.: SEM images of PeGE without (a) and with (b) Cu NPs
The electrochemical measurements were performed and the modified copper nanoparticles
were found to enhance the electrochemical oxidation signal of adenine (Ade) as shown in
Figure 2 compared to the bare electrode.
Figure 2.: Linear sweep voltammograms Ade of 10 µM concentration on PeGE in 0.1 M acetate-phosphate
buffer (pH 7) at a scan rate of 100 mV/s. (a) buffer solution without Ade and Cu NPs; (b) Ade on a bare PeGE,
(c) Ade on copper nanoparticles modified PeGE
4. CONCLUSIONS
The copper nanoparticle-modified pencil graphite electrodes (PeGE) exhibited significantly
enhanced electrochemical signals towards the oxidation of adenine, and a high sensitivity as
well as a wide linear range of electrochemical determination of adenine. The electrochemical
determination proposed here provides a novel biosensing platform for detection of purine
bases.
a b
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5. ACKNOWLEDGEMENTS
This research was supported by the following projects: LH 13053 KONTAKT II (MEYS CR)
the CEITEC – Central European Institute of Technology Project CZ. 1.05/1.1.00/02.0068,
SIX CZ.1.05/2.1.00/03.0072, and the Project Postdoc I, reg. No. CZ.1.07/2.3.00/30.0009. The
project is co-financed by the European Social Fund and the state budget of the Czech
Republic.
6. REFERENCES
[1] Ashihara H., Sano H., Crozier A.: Phytochem., 69 (2008), 841.
[2] Sharma V. K., Jelen F., Trnkova L.: Sensors, 15 (2015), 1564.
[3] Goyal R. N., Gupta V. K., Oyama M., Bachheti N.: Talanta, 71 (2007), 1110.
XV. Workshop of Physical Chemists and Electrochemists
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ATOMIC FORCE MICROSCOPY FOR CHARACTERIZATION
OF BIOMOLECULES, AFFINITY COMPLEXES AND CELLS
Petr SKLÁDAL1,2*
, Jan PŘIBYL1,3
, Veronika HORŇÁKOVÁ1, Patrik GEREG
2, Zdenka
FOHLEROVÁ2, Martin JAKUBEC
2, Zdeněk FARKA
1,2, David KOVÁŘ
1, Martin PEŠL
3
1 Nanobiotechnology, CEITEC, 2
Department of Biochemistry, Faculty of Science, and 3 Department of Biology,
Medical Faculty, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Abstract
The instrumentation and services available at the Core Facility of Nanobiotechnology in
CEITEC MU will be introduced. The research possibilities will be demonstrated on
experiments with proteins, nucleic acids, their affinity complexes and cells. The interaction of
ssDNA binding protein with oligonucleotides was imaged using bare tips (Fig. 1), the binding
forces in the affinity complex were studied using the protein ligand-modified tip and the
ForceRobot AFM head for automated recording of force-distance curves. Similar experiments
characterised immunoreactions between antibody and serum albumin and microbial cells as
antigens and hybridisation of nucleic acids; interactions were confirmed using surface
plasmon resonance and electrochemical measurements. Properties of mast cells were
characterised in relation to the changes accompanying biotransformation events (Fig. 2).
Simultaneous recording of contractions and electric activity of cardiomyocytes was followed
using the AFM cantilever with conductive tip functioning as nanomechanical transducer for
cellular biosensor suitable for evaluation of physiologically active compounds in real time.
XV. Workshop of Physical Chemists and Electrochemists
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Figure 1.: SSB protein binds ssDNA, semicontact
mode, imaged on mica.
Figure 2.: AFM images of mast cells (left)
degranulating after 10 min exposure to the alergen
simulant (right).
ACKNOWLEDGEMENT:
The work was supported by CEITEC – Central European Institute of Technology
(CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.
XV. Workshop of Physical Chemists and Electrochemists
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SWITCHING OF ELECTROCHEMICAL PROPERTIES OF PROTEINS
UPON GLYCATION
Jan VACEK*, Martina ZATLOUKALOVA, Marika HAVLIKOVA, Jitka ULRICHOVA
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University,
Hnevotinska 3, 775 15 Olomouc, Czech Republic
Abstract
In this contribution, a first sensing strategy for protein glycation is proposed, based on protein
electroactivity measurement. Concretely, the label-free method proposed is based on the
application of a constant-current chronopotentiometric stripping analysis at Hg-containing
electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein
signal, peak H, observed at highly negative potentials, which was previously ascribed to a
catalytic hydrogen evolution reaction. Using this method, model water-soluble (bovine serum
albumin, human serum albumin and lysozyme) and poorly water-soluble membrane (Na/K
ATPase) proteins were investigated.
1. INTRODUCTION
Protein glycation is the result of the covalent bonding of the protein molecule with sugars and
their metabolic by-products via a non-enzymatic process. The glycation of proteins occurs by
a complex series of sequential and parallel reactions that form a Schiff’s base, Amadori
products and advanced glycation end-products (AGEs) [1].
In the physiological setting, glucose and other saccharides are important glycation agents, but
the most reactive glycation agents are the α-oxoaldehydes, glyoxal, methylglyoxal and 3-
deoxyglucosone. Methylglyoxal is the most significant glycation agent in vivo, being one of
the most reactive dicarbonyl molecules in living cells. This compound is an unavoidable by-
product of glycolysis. Methylglyoxal irreversibly reacts with amino groups in lipids, nucleic
acids and proteins, forming methylglyoxal-derived advanced glycation end-products
(MAGEs) [1].
Protein glycation is a complex process that plays an important role in diabetes mellitus, aging
and in the regulation of protein function in general. As a result, current methodological
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research on proteins is focused on the development of novel approaches for investigation of
glycation processes.
2. MATERIAL AND METHODS
The samples (I, II and III) were analyzed using a.c. voltammetry (ACV) and constant-current
stripping chronopotentiometric analysis (CPSA). Two types of working electrodes were used,
i.e. hanging mercury drop electrode and silver solid amalgam electrode.
Figure 1: Graphical representation of sample handling and analytical methods used for detecting protein
glycation. Three sample types were investigated in this study: sample I (native protein), sample II (glycated
protein), and sample III, where the glycation process was suppressed with the glycation inhibitor
aminoguanidine (A). All samples were centrifuged (B), supernatants were collected (C), sample III was
subjected to dialysis to eliminate interfering species (D), and finally the acquired supernatants (or dialyzates)
were analyzed. CPSA was used for the analysis, and the results were compared with the results of
complementary methods such as native PAGE, fluorescence spectroscopy and 2D isoelectric
focusing/denaturing PAGE (E).
All measurements were performed at room temperature with a μAutolab III analyzer
(EcoChemie, NL) connected to a VA-Stand 663 (Metrohm, Herisau, Switzerland) in a three-
electrode setup with Ag/AgCl3M KCl and Pt-wire as reference and auxiliary electrodes,
II.
III.
I.
B) Centrifugation
A) Sample Preparation
C) Supernatant CollectionI. II. III.
III.
Methylglyoxal (glycation agent)
Native protein
Aminoguanidine (glycation inhibitor)
PBS
Glycated protein
I. II. III.
D) Dialysis - Interferences
E) Analysis
Electrochemistry Native PAGE Fluorimetry 2D IEF/SDS-PAGE
Sample:
Analytical result:
Native
Glycated
Glycation suppressed
Potential (V) Wavelength (nm)
I.
III.
II.
II.
I.
III.
Elimination
pHI. II. III.
I.
II.
III.
Peak H
II.
III.
I.
B) Centrifugation
A) Sample Preparation
C) Supernatant CollectionI. II. III.
III.
Methylglyoxal (glycation agent)
Native protein
Aminoguanidine (glycation inhibitor)
PBS
Glycated protein
I. II. III.
D) Dialysis - Interferences
E) Analysis
Electrochemistry Native PAGE Fluorimetry 2D IEF/SDS-PAGE
Sample:
Analytical result:
Native
Glycated
Glycation suppressed
Potential (V) Wavelength (nm)
I.
III.
II.
II.
I.
III.
Elimination
pHI. II. III.
I.
II.
III.
Peak H
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respectively. For other details on experimental design and application of complementary
methods see Fig. 1 [2].
3. RESULTS AND DISCUSSION
Here we present a novel electrochemical label-free method for the in vitro monitoring of
protein glycation via observing changes in intrinsic electroactivity of the protein. The study
covers several model protein molecules and methylglyoxal as the glycation agent. The
electrochemical results presented here are supported by previously developed complementary
methods, i.e. native (PAGE) electrophoretic assay, 2D isoelectric focusing-polyacrylamide gel
electrophoresis (SDS-PAGE) and the fluorescence spectroscopy method (Fig. 1) [2].
The general principle of the proposed assay is to monitor an electrocatalytic process called
catalytic hydrogen evolution reaction (CHER), where the protein serves as the catalyst. The
result of the measurement is an electrocatalytic CPS signal observable at negative potentials at
Hg-electrodes known as peak H. Concretely, proton-donating amino acid (aa) residues, Cys
and the basic aa residues Lys, Arg and His, are responsible for the electrocatalytic process, i.e.
CHER [3].
In more detail, the principle of our approach is connected to the covalent modification
(glycation) of the above-mentioned aa residues, because the submolecular targets for non-
enzymatic protein glycation are primarily Lys, Arg, Cys and to a limited extent also His.
Thus, if the glycation reaction proceeds, the electrocatalytically active aa residues are not able
to contribute to the CHER, which is reflected in the changes in peak H in the investigated
protein samples.
4. CONCLUSION
Taking into account the fact that the glycation targets in proteins are the same aa residues as
those participating in the electrocatalytic reaction, we are able to selectively monitor glycation
processes via the decrease in CPS peak H.
5. ACKNOWLEDGEMENT
This work was supported by the Czech Science Foundation (14-08032S, J.V.) and by the
Ministry of Education, Youth and Sports of the Czech Republic (LD14033, J.V. and
CZ.1.07/2.3.00/30.0004, M.H).
XV. Workshop of Physical Chemists and Electrochemists
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6. REFERENCES
[1] Rondeau P., Bourdon E.: Biochimie 93 (2011), 645-658
[2] Havlikova M., Zatloukalova M., Ulrichova J., Dobes P., Vacek, J.: Anal. Chem. 87 (2015), 1757-1763
[3] Palecek E., Tkac J., Bartosik M., Bertok T., Ostatna V., Palecek J.: Chem. Rev. 115 (2015), 2045-2108
XV. Workshop of Physical Chemists and Electrochemists
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TRANSFER OF MONOVALENT IONS IN QUADRUPLEX DNA
SYSTEMS
Matúš DUREC1,2
, Jan NOVOTNÝ1, Petr KULHÁNEK
1,2, Radek MAREK
1,2,3
1 Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
2 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00
Brno, Czech Republic
3 Faculty of Science, Department of Chemistry, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
1. INTRODUCTION
The DNA molecule represents one of the most sophisticated molecular systems. Among the
numerous structural forms of DNA, G-quadruplexes which arise from the self-assembling of
guanine-rich sequences, deserve particular attention. The spatial structure of G-quadruplexes,
governed by non-covalent interactions, can be dissected into three basic organization levels:
a) Assembling of four guanine units into the guanine tetrad (G-tetrad or G-quartet) via
hydrogen bonds; b) Formation of higher-order assemblies (G4)n through π-π stacking
interactions of neighboring G-tetrads; c) Coordination of monovalent ions (M+), which are
typically located in the inter-base regions of the quadruplex channel and impart an additional
stability to the quadruplex structure [1,2].
The design of novel non-natural quadruplexes paves the way for introducing new drugs
through control of several quadruplex-key interactions in which the key can be a wide range
of biomolecules from the quadruplex itself to quadruplex-stabilizing ligands. Modified
quadruplexes can serve not only as cancer-treatment agents, but also as building blocks of
novel biosensors, nucleic acid aptamers, and nanowires [3]. Besides guanine, other purine
bases such as xanthine and its derivatives, are considered to be highly promising building
blocks for designing artificial nucleic acid quadruplexes [4].
In this study, we investigate transfer of monovalent cations (M+) in natural and artificial
quadruplex DNA systems derived from guanine, xanthine, 3-chloro-3-deazaguanine, or 8-
chloro-9-deazaxanthine. Our study further extends models analyzed in previous studies [5–7].
XV. Workshop of Physical Chemists and Electrochemists
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2. RESULTS AND DISCUSSION
Model systems consist of five stacked tetrads and four potassium cations, each in the inter-
tetrad space (see Figure 1). Methods of Density Functional Theory (DFT) were used to
optimize structures of all the systems and to calculate the barriers to ion transfer.
Following the full optimization of (B4)5·M+ systems, the outer tetrads were fixed and systems
re-optimized in the presence of only three M+. Subsequently twenty-one substructures with
systematically modified position of central potassium atom were built, optimized, and their
energies were calculated using DFT and MM methods. The resulting energy profiles will be
presented and discussed in this contribution.
3. ACKNOWLEDGMENT
This work was supported by the project “CEITEC – the Central European Institute of
Technology” (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.
Computational resources were provided by the MetaCentrum under the program LM2010005
and the CERIT-SC under the program Centre CERIT Scientific Cloud, part of the Operational
Program Research and Development for Innovations, Reg. no. CZ.1.05/3.2.00/08.0144.
Figure 1: Model systems consisting of five stacked tetrads and four monovalent cations employed in this
study.
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4. REFERENCES
[1] Yurenko Y. P., Novotný J., Mitoraj M. P., Sklenář V., Michalak A., Marek R.: Journal of Chemical Theory
and Computation, 10 (2014), 5353–5365.
[2] Yurenko Y. P., Novotný J., Sklenář V., Marek R.: Physical Chemistry Chemical Physics, 16 (2014), 2072–
2084.
[3] Davis J. T., Angewandte Chemie International Edition, 43 (2004), 668–698.
[4] Bazzi S., Novotný J., Yurenko Y. P., Marek R.: Chemistry – A European Journal, 21 (2015), in press.
[5] van Mourik T., Dingley A. J.: Chemistry – A European Journal, 11 (2005), 6064–6079.
[6] Novotný J., Yurenko Y. P., Kulhánek P., Marek R.: Physical Chemistry Chemical Physics, 16 (2014),
15241–15248.
[7] Gkionis K., Kruse H., Platts J. A., Mládek A., Koča J., Šponer J.: Journal of Chemical Theory and
Computation, 10 (2014), 1326–1340.
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DETECTION OF GLUCOSE USING GEL-TEMPLATED GOLD
NANOSTRUCTURED ELECTRODES
Tomáš JUŘÍK1*
, Zdeněk FARKA1, David KOVÁŘ
1, Pavel PODEŠVA
2, František FORET
1,2,
Petr SKLÁDAL1,3
1 CEITEC MU, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
2 Institute of Analytical Chemistry AS CR, Veveří 97, 602 00 Brno, Czech Republic
3 Department of Biochemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech
Republic
Abstract
A precise monitoring of glucose level in blood is of high importance in clinical medicine,
thus, sensitive, rapid and reliable methods for its detection are required. In this work, gelatin
templated gold nanostructures were fabricated in order to improve the sensitivity of glucose
analysis. SEM and AFM were used for characterization of the created surfaces. Glucose was
detected by a direct electrochemical oxidation during cyclic voltammetry in alkaline solution.
Limit of detection of 10 μM was achieved in aqueous samples. The sensor was also able to
detect real concentration of glucose in deproteinised human serum with negligible effect of
interferents. All results were verified by commercial glucometer and the standard kit for
photometric detection of glucose.
1. INTRODUCTION
Nanoscopic surfaces impose very high electroactive area, conductivity and small electrolyte
diffusion resistance. The electrodes with fabricated nanostructures represent a powerful tool
regarding to the acceleration of surface bound reactions with sluggish kinetics, such as
glucose electrooxidation. The mechanism of electrooxidation includes a two-step reaction;
glucose becomes oxidised in the forward scan of cyclic voltammetry by the catalysis of OH-,
this is followed by the second oxidation step in the backward scan catalysed by O2-
created
during the reduction of gold oxide. Herein, we introduce a non-enzymatic nanostructured
sensor with high sensitivity and long-term stability for rapid detection of glucose in alkaline
media and human blood samples.
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2. MATERIAL AND METHODS
Preparation of electrodes
A gold layer sputtered on a glass wafer with chromium adhesion layer was used as a support.
A specific pattern of the chip was designed by photolithographic technology leaving a circular
area for the electrodeposition of gelatin / agarose templated gold nanostructures. The resulting
morphology was dependent on the time of electrodeposition in electroplating bath, current
density and composition of the gel. Cyclic voltammetry in 1 mM ferro/ferricyanide was used
for characterisation of new surfaces. Nanostructured surfaces were visualised by SEM and
AFM.
3. RESULTS AND DISCUSSION
Surfaces characterisation
During the electroplating, gold nanostructures grow through the gelatin pores and forms thin
plates with length of 1 µm and width around 50 nm. Several sawtooth-like objects with sharp
tips (diameter of 10 nm) can be observed on each plate (Fig. 1). This provides surface with an
electroactive surface area (ESA) magnified by 60 folds and high roughness factor (16.7)
exhibiting a remarkable electrocatalytic activity towards electrooxidation of glucose.
Figure 1: SEM image of gelatin templated gold nanostructures.
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Determination of glucose
Glucose was analysed in 50 mM KOH. Two peaks corresponding to double oxidation of
glucose are evident in Fig. 2. A broad linear range for the detection of glucose from 32 µM to
10 mM was achieved. The detection limit was estimated to be 90 µM and 10 µM in case of
cyclic voltammetry and amperometry, respectively.
Figure 2: Cyclic voltammetry of different concentrations of glucose in 50 mM KOH.
The concentration of glucose was determined in deproteinised and alkalised standard human
blood sera and real blood samples. No significant interference of other blood species was
observed. All results were evaluated from the peak of the backward scan by the standard
addition method and compared with verified methods. The determined concentration
(1.3 ± 0.2 mM) correlated with the results measured by glucometer and photometric methods.
The factor of dilution was 4, i.e. the real concentration corresponded to 5.2 mM. After
cleaning, the long-term stability of the sensor allows reproducible detection of glucose.
4. CONCLUSION
In this study, a non-enzymatic nanostructured sensor for a sensitive detection of glucose was
developed. An extensive magnification of the electroactive surface provided excellent
conditions for the glucose oxidation with limit of detection 10 μM. A low operational
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potential enabled glucose analyses in human blood samples without interference of other
oxidisable blood components. The sensor stability and reproducibility make it promising for
future applications in clinical analytics.
5. ACKNOWLEDGEMENT
The work has been supported by CEITEC – Central European Institute of Technology
(CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.
6. REFERENCES
[1] Toghill K E, Compton R G: Int. J. Electrochem. Sci. 5 (2010), 1246–1301
[2] Pasta M, La Mantia F, Cui Y: Electrochim. Acta. 55 (2010), 5561–5568
[3] Wang J, Cao X, Wang X, Yang S, Wang R: Electrochim. Acta. 138 (2014), 174–186
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ELECTROCHEMICAL CORROSION OF STEEL AS A SOURCE
OF FE2+
CATALYST OF FENTON REACTION
Veronika KOČANOVÁ*, Libor DUŠEK
Institute of Environmental and Chemical Engineering, Faculty of Chemical Technology, University of Pardubice,
Studentská 573, 532 10 Pardubice, Czech Republic
Abstract
Even if Fenton reaction is well known for years, it can be considered as an efficient
and perspective method of wastewater treatment. Commonly used catalysts of Fenton
oxidation are ions of Fe2+
. Catalyst in form of Fe2+
is meeting important attributes and can be
used on various organic pollutions [1]. Highest efficiency is achieved when pH value is 2,8
so experiments were processed with pH index range from 2 to 4 [2]. A way of dosing catalyst
for electrochemical dissolving of sacrificial steel anode was chosen. Two types of steel – alloy
steel Cr-Ni and non-alloy steel were used as a source of Fe2+
ions. The influence of current
density for corrosion loss according to used material of anode was observed. The usage of Cr-
Ni steel is five times more expensive but can be taken as a well regulated source of Fe2+
ions.
Non-alloy steel shows high material yield even with low current density, but the level
of regulation of Fenton resp. electro-Fenton process is low.
1. INTRODUCTION
Environmental technologies, which are based on Fenton resp. electron-Fenton oxidation
are commonly used in terms of wastewater treatment. Ordinary catalyst of Fenton processes
are ions of Fe2+
. It is relatively cheap, non-toxic, regenerable and easy to be removed
from treated water source of Fe2+
ions. As an efficient way of dosing catalyst, electrochemical
dissolving of sacrificial steel anode can be chosen.
2. MATERIAL AND METHODS
Steel 17 240 Cr-Ni (DIN X 5 CrNi 18 10, AISI 304) and Steel 11 373 (DIN USt 37-2) were
used experimentally as a sacrificial steel anodes. Cathode was made from Platinum. Tests
were processed with pH index 2, 3 and 4. Constant value of pH was maintained thanks
to automatic titrator TitraLab 856 (Radiometer analytical, Lyon, France). For each level
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of pH, amount of current was 10 mA, 25 mA, 50 mA, 100 mA and 150 mA. These current
levels were set on DC Power Supply SDP – 2210 (Manson, Kwai Chung, N.T., Hong Kong).
Concentration of Fe2+
was found out by values of absorbance, which we gauged every 15
minutes since creation of color complex. For assessment of reaction with 1,10-phenanthroline,
spectrophotometric method was used. For measurement, was used UV/VIS
Spectrophotometer Libra S22 (Biochrom, Cambridge, United Kingdom). Total iron was
observed gravimetrically – steel anode was continuously weighted up on analytical weights –
Digital analytical balance 870 (Kern, Balingen, Germany)
3. RESULTS AND DISCUSSION
Setup of electrochemical dissolving terms was always identical for both of those used
materials of sacrificial steel anode. Experiments were run at currents 10 mA, 25 mA, 50 mA,
100 mA, 150 mA and at constant pH 3. Collection of sample for determination of Fe2+
was
carried out always at the same time during the experiment. Weight loss of sacrificial anode
was found at the same time simultaneously. Because concentrations of Fe2+
were very low
in comparison of total iron dependence concentration of Fe2+
on time summarize next Graph
1. and 2.
Figure 1.: Concentration of Fe2+(Steel ČSN 17 240), pH 3 Figure 2.:Concentration of Fe2+(Steel ČSN 11373),pH 3
The following Graph 3. and 4. shows weight losses of iron anode during the duration
of experiment.
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Figure 3.: Weight losses of Fe (Steel ČSN 17 240), pH 3 Figure 4.: Weight losses of Fe (Steel ČSN 11373), pH 3
Also corrosion tests were carried out without electric current. The experiment shows
that the Steel 17 240 Cr-Ni without current almost not dissolve. It can be concluded that
the influence of atmospheric oxygen is negligible. The corrosion rates of Steel 17 240 Cr-Ni
are thousand times lower than when we used Steel 11 373 in pH 3. And the corrosion losses
of Steel 17 240 Cr-Ni are eight thousand times lower than when we used Steel 11 373 in pH
3. Ferrous and ferric sludge could be removed by sedimentation. At first solutions were
neutralized. Then ions were precipitated and then sedimented. Collected samples met
the Government Regulation about values of pollution by iron in the surface and wastewaters.
Limit for iron is 5 mg·l-1
. Measured values of absorbance and concentrations are shown
in the following Table 1.
Table 1: Measured values of concentrations electrochemically dissolved Fe2+
and Fe3+
ions in a liquid
portion after the precipitation and sedimentation
pH absorbance
Fe2+
[-]
absorbance
Fe [-]
conc. Fe2+
[mg∙l-1
]
conc. Fe3+
[mg∙l-1
]
conc. Fe
[mg∙l-1
]
1. 7 0,074 0,091 0,15 0,23 0,38
2. 10,5 0,075 0,089 0,16 0,22 0,37
4. CONCLUSION
Fenton, resp. electro-Fenton oxidation are perspective environmental technologies for waste
water treatment. Ferrous catalyst was used because it is efficient, cheap, non- toxic, well
regenerable and removable. Effective method of catalyst dosage is the electrochemical
dissolution of sacrificial steel anode. It was comparison between two types of material – alloy
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steel and non-alloy steel. Ferrous and ferric sludge could be effectively removed
by sedimentation.
5. ACKNOWLEDGEMENT
This work was supported by the Ministry of Education, Youth and Sports of the Czech
Republic (project No. SG 350006).
6. REFERENCES
[1] Nidheesh P. V., Gandhimathi R.: Desalination, 299, (2012), 1-15
[2] Brillas E., Sires I., Oturan M. A.: Chem. Rev., 109, (2009), 6570-6631
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DEVELOPMENT OF FLUORESCENT SUBSTRATES OF
HALOALKANE DEHALOGENASES FOR MECHANISTIC
ENZYMOLOGY
Zuzana KORENČIAKOVÁ1, David BEDNÁŘ
1, Jan BREZOVSKÝ
1, Petr KLÁN
3,
Jiří DAMBORSKÝ1,2
, Zbyněk PROKOP1,2*
1 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in
the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech
Republic
2 International Centre for Clinical Research, St. Anne's University Hospital, Pekarska 53, 656 91 Brno, Czech
Republic
3Department of Chemistry and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of
Science, Masaryk University, Kamenice 5/A7, 625 00 Brno, Czech Republic
Abstract
Haloalkane dehalogenases (HLDs) are α/β hydrolases, which catalyze hydrolytic
dehalogenation reaction and can be used in multiple applications including bioremediation of
environmental pollutants and organic synthesis [1]. The complexity of their structure and
reaction make them suitable targets for mechanistic studies.
Fluorescent substrates provide a useful tool for detailed characterization of enzymatic
reactions even at a single-molecule level. However, the amount of such substrates is limited
and available only for a small number of enzymes. First fluorescent substrates of HLDs were
discovered by molecular docking [2]. Following preliminary experiments with 4
coumarin-based dyes were conducted identifying a fluorescent substrate, which had been
proven to provide a spectral change during the reaction catalyzed by DmmA dehalogenase.
In the first part of this project, activity assay employing coumarin-based substrate was
optimized, enabling the collection of valuable kinetic data with the enzyme DmmA, including
the lowest Michaelis-Menten constant ever observed for HLDs. Moreover, systematic activity
measurements of seven selected HLDs towards the fluorescent substrate demonstrated the
broad applicability of the novel fluorescence assay.
In the second part of the project, docking of chosen organic fluorophores produced
energetically favourable binding modes with potential for SN2 substitution. Fifteen molecules
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were bound into four different enzymes with potentially reactive geometry. Selected
molecules will be synthetized and experimentally tested.
REFERENCES
[1] T. Koudelakova, S. Bidmanova, P. Dvorak, et al.: Biotechnology Journal, 8 (2013), 1, 32-45
[2] L. Daniel, T. Buryska, Z. Prokop, et al.: Journal of Chemical Information and Modeling, 55 (2015), 1,
54-62
[3] L. Michaelis and M. L. Menten: Biochemische Zeitschrift, 49 (1913), 333-369
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ANALYSIS OF BIODEGRADABLE NANOFIBROUS LAYERS
A. KOTZIANOVÁ1,2*
, J. ŘEBÍČEK2, M. POKORNÝ
2, J. HRBÁČ
1, V. VELEBNÝ
2
1 Masaryk University, Faculty of Science, Department of Chemistry, Kamenice 5, CZ-62500 Brno, CZE
2 Contipro Biotech s.r.o., R&D Department, CZ-56102 Dolni Dobrouc, CZE
Abstract
A method for the determination of nanofibrous mats chemical composition based on Raman
spectroscopy and singular value decomposition is presented. Various composite samples
consisting of polycaprolactone (PCL), poly(ethylene) oxide (PEO) and hyaluronic acid (HA)
were prepared by electrospinning. Using confocal Raman spectroscopy, we were to
distinguish substantial changes in the distribution of the polymers caused by various
preparation parameters. The ratio between both polymers was expressed as the relative
fraction of the particular chemical compound.
1. INTRODUCTION
The method known as electrospinning attracts many research groups all over the world
especially because of its simplicity, versatility and efficiency in producing nanofibrous
materials. Electrospinning (ES) uses electrostatic forces to produce nanofibrous layers from
a polymer solution. As prepared nanofibrous materials have different properties than bulk
materials, such as very high porosity or huge surface-to-volume ratio, there is a potential for
their application in many fields (health care, environment, textile and chemical industry or
electronics). Our work focuses on the preparation and characterization of polymeric
nanofibrous layers for use especially in the biomedical field [1].
Using our laboratory electrospinning device, we prepared samples made of PEO, PCL and
HA. The prepared layers were analysed using a method based on Raman spectroscopy and
Singular Value Decomposition [2]. The characterization of produced materials could greatly
benefit the optimization of the electrospinning process.
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2. MATERIAL AND METHODS
Materials
The HA/PEO nanofibrous layers were prepared using a 6% w/w blend of poly(ethylene) oxide
(PEO, 600 kDa, SigmaeAldrich) and hyaluronic acid (HyA, 82 kDA, Contipro Pharma a.s.)
dissolved in distilled water. The HA/PCL nanofibrous layers were prepared using a 13% w/w
blend of polycaprolactone (PCL, 80 kDa, SigmaeAldrich) and hyaluronic acid (HyA, 82 kDA,
Contipro Pharma a.s.) dissolved in chloroform:methanol (1:1).
Methods
The commercially available laboratory device 4SPIN® LAB1 (www.4spin.info) was used for
electrospinng. Each sample was produced from a single solution of HA and PEO or PCL
blend. Thus there were solutions to be spun with three different ratios between HA and PEO -
1:4, 1:1 and 4:1 and with three different ratios between HA and PCL – 1:2, 1:1 and 2:1.
An in-house developed Raman system consisting of a confocal probe (own design, [3])
connected via an optical fibre to a dispersive spectrograph equipped with a multichannel CCD
detector. Raman scattering was excited by a 632.8 nm line (13 mW at the sample) of a He-Ne
laser. Advanced multivariate procedures based on singular value decomposition (SVD) were
applied in data treatment and spectral analysis of the chemical composition of the samples.
3. RESULTS AND DISCUSSION
Figure 1 shows SEM images and Raman spectra of the HA:PEO nanofibres. The amount of
HA increases from sample 1 to sample 3. Different ratios between HA and PEO in samples
were confirmed by RS; intensity changes in the HA and PEO bands can be observed. The
SVD analysis plot shows homogenous distribution of HA:PEO within each sample.
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Figure 1.: SEM images, Raman spectra and results of SVD analysis of the HA:PEO nanofibers.
Figure 2 shows large variations in the distribution of HA:PCL within each prepared sample,
which is why the points in the SVD analysis plot are scattered all over. The Raman spectra
differ within each sample and it is impossible to observe a sequential increase of HA content
as was intended. The inhomogeneity of the samples was caused by insufficient mixing of HA
and PCL in the solution.
Figure 2.: SEM images, Raman spectra and results of SVD analysis of the HA:PCL nanofibers
4. CONCLUSION
Using RS it was possible to non-destructively show the inhomogeneity of the samples
prepared via electrospinning. Although each sample was prepared from a single solution of
polymers and solvents, the quality of the resulting nanofibrous products was influenced by the
stirring process. As both HA and PEO are soluble in water, the produced nanofibrous layers
are homogenous. On the other hand, PCL is not soluble in water and it is necessary to use an
organic solvent to prepare a HA:PCL solution for the spinning process. This leads to poor
mixing of HA and PCL, resulting in an inhomogeneous distribution of fibres throughout the
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sample. Results show that the combination of RS and SVD allows to control the nanofiber
spinning process and thus to produce homogenous nanofibrous layers.
5. ACKNOWLEDGEMENT
This research project was partly conducted under financial support provided by the
Technology Agency of the Czech Republic (project TA02011238: Novel wound dressings
based on nanofibers and staple microfibers of hyaluronan and chitin/chitosan-glucan
complex).
6. REFERENCES
[1] Kotzianova A., et al. : Polymer, 55 (2014), 5036-5042
[2] Palacky J., et al. : J Raman Spectrosc, 42 (2011), 1528-39
[3] Pokorny M., et al.: Contipro Biotech s.r.o., patent CZ304711B6
[4] Siesler H.W., et al.: Infrared and Raman spectroscopy of polymers, 1980
[5] Seidel A.: Characterization and Analysis of Polymers, Wiley, 2008
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ELECTROCHEMICAL LI INSERTION INTO TIO2 POLYMORPHS:
STUDY OF MECHANISM AND STRUCTURAL CHANGES
Barbora LASKOVA1,2
, Marketa ZUKALOVA1, Otakar FRANK
1, Milan BOUSA
1,2 and
Ladislav KAVAN1,2
1 Department of Electrochemical Materials, J. Heyrovsky Institute of Physical Chemistry of the ASCR, v. v. i.,
Dolejskova 2155/3, CZ-182 23 Prague 8, Czech Republic
2 Department of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, CZ-128 43
Prague 2, Czech Republic
Abstract
The lithium insetion into TiO2 anatase and TiO2(B) was studied by electrochemical and
spectroelectrochemical methods. The electrochemical data were analysed by cyclic
voltammogram deconvolution and the capacitive contribution in TiO2(B) was higher by about
30% compared to that in anatase despite of smaller surface area of the former. The difference
indicates pseudocapacitive Li-storage in the bulk TiO2(B). The detailed
spectroelectrochemical study of Li-insertion into anatase for four isotopologue combinations
in the system, namely 6/7
LixTi16/18
O2 , enabled improved spectral assignment of this structure
in Raman spectroscopy.
1. INTRODUCTION
Titanium dioxide is a widely studied material due to its attractivity for many applications as
photoelectrochemical solar cells, photocatalysis and Li-ion batteries. Especially two titanium
dioxide polymorphs, namely the tetragonal TiO2 anatase and monoclinic TiO2(B), are very
promissing for application in Li-ion batteries. Therefore the electrochemical insertion of
lithium into these structures was intensively studied during last dacades[1-3]. However, there
are still open questions about the mechanism and structural changes during Li insertion into
TiO2(B) and anatase. Hence we investigated the differences in Li-insertion into these two
materials by the method of elctrochemical cyclic voltammogram deconvolution [4] and we
studied the structural changes in anatase during lithium insertion by Raman
spectroelectrochemistry using oxygen/lithium isotope labeled TiO2/electrolyte [5].
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2. MATERIALS AND METHODS
TiO2, Ti18
O2 anatase were synthesized using a method described in [4,5]. Briefly, TiCl4 was
mixed with H218
O (Aldrich 18
O 99%) or with ordinary water (H216
O). The mixture was then
heated in vacuum at 450°C to create tetragonal anatase phase of TiO2. The synthetic protocol
for TiO2(B) is described elsewhere.[4] The lithium-isotope labeled salt 6LiClO4 (with 95 atom
% of 6Li, Sigma Aldrich) was used as electrolyte in some spectroelectrochemical
measurements [5].
The preparation of electrodes and the used methods are reported elsewere [4,5]. The details of
set-up used for electrochemical measurement are described in [4]. The spectroelectrochemical
cell assembling and Raman spectroelectrochemical method are presented in [5].
3. RESULTS AND DISCUSSION
The cyclic voltammetry (CV) was applied on phase pure TiO2 anatase and TiO2(B) in a range
of potentials 1.3-2.5 V against the Li/Li+ reference electrode. Subsequently, the resulting
voltammograms were analyzed by the method of cyclic voltammogram deconvolution, which
was firstly reported by Dunn et al.[6,7] On the base of this method the current response at a
fixed potential can be expressed by i(V)= k1ν +k2v1/2
, where k1ν corresponds to the capacitive
current contribution associated with the storage of Li+ at the TiO2 surface and the k2v
1/2
corresponds to the diffusion-controlled current, which is attributed to the insertion of Li+ in
the bulk of TiO2 lattice. After rearrangement of the equation above it is possible to determine
the coefficients k1 and k2 from measured data and to compute the capacitive and diffusion
contributions to the total current for each potential at CV. The computed capacitive charge
storage contribution to the total stored charge at scan rate 0.5mVs-1
is 68% and 37% for
TiO2(B) and TiO2 anatase, respectively. The TiO2(B) has obviously larger capacitive
contribution (by about 30%) compared to that in anatase, in spite of the factor of 3 smaller
surface area of the former (see [4]). The explanation of this difference could be a monoclic
structure of TiO2(B) with open channels along the b-axis. The data indicate a pseudocapacive
Li+ storage in these TiO2(B) channels which causes higher capacitive contribution to the total
current response compared to anatase. For more details see [4].
The Li+ insertion into anatase was also studied by Raman spectroelectrochemistry employing
four different isotopologue combinations in the system, namely 6/7
LixTi16/18
O2 . During Li+
insertion the tetragonal anatase phase is transformed to orthorhombic structure LixTiO2, where
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x is the insertion coefficient. Using of different isotopologues in the system caused the shifts
of Raman peaks corresponding to the vibration employing the used isotope. Therefore this
technique is a really useful tool for assignment of the observed peaks in Raman spectra to the
corresponding vibrational modes in structure. On the basis of these techniques and DFT
calculations we were able to assign all the observed 20 modes (out of 42 modes theoretically
expected) in the Raman spectrum of LixTiO2. For more details see [5].
4. CONCLUSION
The electrochemical and spectrolectrochemical investigation of TiO2 anatase and TiO2(B) was
carried out. The cyclic voltammograms of Li+ insertion into anatase and TiO2(B) were
analyzed by voltammogram deconvolution and the capacitive contribution in TiO2 (B) was
higher by about 30% compared to that in anatase despite of smaller surface area of the former.
These data indicate pseudocapacitive Li-storage in the TiO2 (B) channels.[4] Subsequently the
structural changes in anatase lattice during Li insertion were studied by Raman
spectroelectrochemistry. The spectroelectrochemical study was performed on four
isotopologue combinations in the system, namely 6/7
LixTi16/18
O2 and all the observed 20
modes in Raman spectrum of LixTiO2 were assigned [5].
5. ACKNOWLEDGEMENT
The work was supported by the Grant Agency of the Czech Republic (contracts No. 13-
07724S and 15-06511S).
6. REFERENCES
[1] Kavan L, Gratzel M, Gilbert S E, Klemenz C, Scheel H J : J.Am. Chem. Soc. , 118 (1996) , 6716-6723
[2] Kavan L Grätzel M, Rathousky J, Zukal A : J. Electrochem. Soc. 143 (1996), 394-400
[3] Zukalova M, Kalbac M, Kavan L, Exnar I, Grätzel M: Chem. Mater. 17 (2005), 1248-1255
[4] Laskova B, Zukalova M, Zukal A, Bousa M, Kavam L: J. Power Sources 246 (2014), 103-109
[5] Laskova B, Frank O, Zukalova M, Bousa M, Dracinsky M, Kavan L: Chem. Mater. 25 (2013), 3710−3717
[6] Wang J, Polleux J, Lim J, Dunn B: J. Phys. Chem. C 111 (2007), 14925-14931
[7] Brezesinski T., Wang J, Polleux J, Dunn B, Tolbert S H: J. Am. Chem. Soc. 131 (2009), 1802-1809.
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VOLTAMMETRIC BEHAVIOUR OF HERBICIDE LINURON ON
BORON-DOPED DIAMOND ELECTRODE
Michaela ŠTĚPÁNKOVÁ*, Renáta ŠELEŠOVSKÁ, Lenka JANÍKOVÁ,
Jaromíra CHÝLKOVÁ
Institute of Environmental and Chemical Engineering, Faculty of Chemical Technology, University of Pardubice,
Studentská 573, 532 10 Pardubice, Czech Republic
Abstract
The possibility of application of the boron-doped diamond electrode (BDDE) has been
investigated for voltammetric analysis of herbicide linuron. Various voltammetric methods
like cyclic voltammetry (CV), direct current voltammetry (DCV) and differential pulse
voltammetry (DPV) were examined. DPV was applied for determination of linuron in model
solutions. Finally, the proposed method was applied for the analysis of spiked river water
sample.
1. INTRODUCTION
Linuron (LIN, 3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea (IUPAC), CAS: 330-55-2) is
a substituted urea pre- and post-emergence herbicide. It is used to control perennial and
annual broadleaf and grassy weeds on both crop and non-crop sites. It is labeled for field and
storehouse usage in such crops as soybean, potato, cotton, bean, com, pea, winter wheat,
carrot, asparagus and fruit crops. LIN is a slightly toxic compound and belongs in EPA
toxicity class III [1, 2]. BDDE corresponds with the concept of green analytical chemistry.
The great advantage of this electrode is a wide potential window. The other important
properties of BDDE are high hardness and chemical inertness, extreme electro-chemical
stability, high thermal stability and stable background current [3]. This electrode was used for
determination of various pesticides up to now [4, 5]. The voltammetric behaviour of LIN on
BDDE is described in the present paper.
2. MATERIAL AND METHODS
All measurements were provided by computer controlled Eco-Tribo Polarograph (Polaro-
Sensors, Praha, Czech Republic) equipped by POLAR.PRO 5.1software for Windows. in 3-
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electrodes set up. BDDE was used as a working electrode (Windsor Scientific Ltd, United
Kingdom), saturated argentchloride as a reference and platinum wire as an auxiliary electrode
(both Monokrystaly, Turnov, Czech Republic). All chemicals used for the preparing of the
standard solutions, electrolytes and other stock solutions were of p.a. purity. Britton-Robinson
(B-R) buffer of a pH value from 2 to 12 was prepared from an alkaline component of 0.2 M
NaOH (Lachema, Brno, Czech Republic) and an acidic component consisting of 0.04 M
H3PO4, 0.04 M H3BO3 and 0.04 M CH3COOH (Lachema, Brno, Czech Republic). 1×10-3
M
stock solution of LIN (purity 99.7 %, Sigma Aldrich, Praha, Czech Republic) was prepared by
dissolution in 70 % acetonitrile and stored in the refrigerator.
3. RESULTS AND DISCUSSION
In this study the BDDE was used to investigate the electrochemical behaviour of LIN. Using
CV it was found that LIN provides 1 irreversible oxidation peak at about 1250 mV in wide
range of pH. B-R buffer of pH 2 served as a supporting electrolyte because the highest
oxidation response was observed in this media. The linear dependence of peak height and the
square root of scan rate was measured using DCV and it corresponds to the diffusion-
controlled electrode process. The DPV technique with optimized parameters was proposed as
a suitable method for LIN determination. Some statistical parameters were obtained. The
linear dynamic range (LDR) was found from 5×10−7
to 1.2×10−4
M. The relative standard
deviation of 11 repeated measurements was calculated as RSDM(11) = 0.84 %. The values of
relative standard deviations of repeated determinations for various concentration levels were
calculated and the results summarized in Table 1 prove very good repeatability of applied
method. Limit of detection (LOD) for LIN was achieved as 1.41×10−7
M. The example of
concentration dependence is shown in Figure 1. The applicability of the BDDE for DPV
determination of LIN was verified by analysis of real sample of spiked river water.
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Table 1: Relative standard deviations of repeated determinations obtained on BDDE.
Added [mol L-1
] Found [mol L-1
] RSDs (5) [%]
1.00 10−5
(1.02 0.010) 10−5
1.49
5.00 10−6
(5.00 0.012) 10−6
0.36
2.50 10−6
(2.50 0.015) 10−6
0.91
Figure 1.: The concentration dependence of LIN obtained on BDDE. Method – DPV, electrolyte – B-R buffer
(pH 2), Ein = 400 mV, Efin = 1600 mV, v = 50 mV s-1
, pulse height 70 mV, pulse width 20 ms, cLIN = 5×10−6
–
4.5×10−5
M.
4. CONCLUSION
The voltammetric behaviour of herbicide linuron on boron-doped diamond electrode was
investigated and a novel analytical method was developed for its determination. DPV in
combination with BDDE was successfully applied for determination of the herbicide in spiked
river water. It can be concluded that the proposed method can be considered as a sensitive and
environmentally acceptable tool for LIN analysis.
400
800
1200
1600
2000
600 800 1000 1200 1400
I[n
A]
E [mV]
Ip [nA] = 30.508 0,377 c [µmol L-1] + 38.747 10.603R² = 0.9989
0
400
800
1200
1600
0 10 20 30 40 50
I p[n
A]
c [µmol L-1]
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5. ACKNOWLEDGEMENT
This work was supported by the University of Pardubice (project No. SGSFChT_2015006)
and by The Ministry of Education, Youth and Sports of the Czech Republic (project No.
CZ.1.07/2.3.00/30.0021).
6. REFERENCES
[1] [online]. [cit. 2015-03-06]. Dostupné z: http://extoxnet.orst.edu/pips/linuron.htm
[2] [online]. [cit. 2015-03-06]. Dostupné z: http://pmep.cce.cornell.edu/profiles/extoxnet/haloxyfop-methylpara
thion/linuron-ext.html
[3] Musilová J, Barek J, Pecková K: Chemické Listy, 103 (2009), 469-478
[4] Bandžuchová L, Švorc L, Vojs M, et all.: Electrochimica Acta, 111 (2013), 242-249
[5] Šelešovská R, Janíková L, Chýlková J: Monatshefte für Chemie - Chemical Monthly, in press (2014), DOI
10.1007/s00706-014-1372-9)
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DESIGNING NUCLEOBASES FOR NUCLEIC ACID QUADRUPLEXES
Yevgen YURENKO1, Jan NOVOTNY, Sophia BAZZI, Radek MAREK
1,2*
1 Central European Institute of Technology, Masaryk University, Kamenice 5, CZ – 62500 Brno, Czech Republic.
E-mail: [email protected]
2 National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500
Brno, Czech Republic
Abstract
This contribution reports in silico design of artificial building blocks for nucleic acid
quadruplexes derived from guanine and xanthine. The results suggest that that the 3-halo-3-
deazaguanine and 9-deaza-8-haloxanthine bases are highly promising candidates for the
development of artificial quadruplexes and quadruplex-active ligands.
1. INTRODUCTION
Chemical alterations of nucleic acid quadruplexes have been subject of numerous studies not
only for their paramount biological importance, but also due to the wide range of potential
applications ranging from drug design and medicinal chemistry to nanosciences and
functional materials. It is known that besides guanine and its derivatives, other purine bases
can form quadruplex structures. Among these bases, xanthine (Xan) considered as one of the
most promising scaffolds for construction of artificial DNA quadruplexes due to its ability to
form very stable tetrads and favorably interact with metal ions (Na+, K
+) located inside
quadruplex channels. In this study, we introduce a new class of quadruplex nucleobases
derived from guanine and xanthine designed for various applications in smart quadruplex
ligands as well as quadruplex-based aptamers, receptors, and sensors.
2. MATERIALS AND METHODS
The formation of quadruplexes was studied in three steps, i.e., formation of base tetrads
specified as (B4) (B = nucleobase), two tetrads stacked on top of each other (B4)2, two-
stacked complexes with a metal cation (K+/Na
+) located in their central cavity (B4)2·M
+ and
three-stacked systems (B4)2·2M+. The formation energies were calculated at the BLYP-
D3/def2-TZVPP level of theory. Then the models of parallel-stranded modified quadruplexes
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were simulated in an explicit solvent using the AMBER12 package and modified AMBER
force fields.
3. RESULTS AND DISCUSSION
The analysis of formation energies of tetrads, as well as stacks of two or three tetrads with
coordinated Na+/K
+ ions suggests that 3-halo-3-deazaguanine and 9-deaza-8-haloxanthine
bases form the most stable quadruplexes with increased contribution from three major non-
covalent interactions (H-bonding, π-π stacking and ion coordination, Figure 1) as compared to
systems from unmodified guanine and xanthine.
Figure 1. The graphical representation of three major non-covalent interactions (H-bonding, stacking and ion
coordination) in quadruplexes with 9-deaza-8-haloxanthine bases. The graph (in the center) shows the stacking
energy for systems with different halogen atoms (F, Cl, Br, I).
The results of molecular dynamics simulations in explicit solvent indicate that quadruplexes
with 3-halo-3-deazaguanine and 9-deaza-8-haloxanthine bases remain stable in solution and
the modifications do not involve serious steric clashes.
4. CONCLUSION
We developed a computational strategy of the rational modification of the quadruplex core to
obtain DNA quadruplexes and quadruplex-based ligands with a higher stability. The results
evidence that 3-halo-3-deazaguanine and 9-deaza-8-haloxanthine bases are promising
scaffolds for construction of artificial quadruplexes and quadruplex-based ligands.
5. ACKNOWLEDGEMENT
The work has been supported by INBIOR (CZ.1.07/2.3.00/20.0042) project.
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6. REFERENCES
[1] Novotný J, Kulhánek P, Marek R: J. Phys. Chem. Lett, 3 (2012), 13, 1788-1792.
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THE COMPARISON OF CHEMICAL AND MAGNETIC
CHANNELRHODOPSIN-2 TRANSFECTION EFFICIENCY
Larisa BAIAZITOVA1*
, Ondřej SVOBODA1,2
, Vratislav ČMIEL1,3
, Ivo PROVAZNÍK1,3
,
Zdenka FOHLEROVÁ2, Jaromír HUBÁLEK
2,4
1 Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno
University of Technology, Technicka 3082/12, 616 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
3 International Clinical Research Center - Center of Biomedical Engineering, St. Anne's University Hospital
Brno, Brno, Czech Republic
4 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of
Technology, Technicka 3082/12, 616 00 Brno, Czech Republic
Abstract
In this work the chemical (polyethylenimine) and magnetic nanoparticle (MATRA) based
transfection methods of channelrhodopsin-2 to HEK293 cell has been compared. We
optimized transfection protocol by changing reagent and DNA amounts and the most
appropriate fluorescent results were obtained after 24 hours of incubation from transfection
time.
1. INTRODUCTION
HEK293 cells has been used in our experiment. This cell line was originally isolated from
primary human embryonic kidney cells transformed by sheared adenovirus 5 DNA in 1970s
[1]. Currently, HEK293 cell line is widely used in stably transfected forms due to such
properties as its quick and easy cultivation and maintenance, easy transfection and processing
of proteins [2].
Transfection is the process of introducing nucleic acids into cells. The transfection techniques
can be classified into the biological, chemical and physical methods [3]. Also the transfection
approach can be classified in two general types based on the DNA expression stability: i)
transient transfection (DNA is expressed only for a limited time and is not integrated into the
genome) or ii) stable transfection (whether the DNA persists in the cells long-term and is
passed to the progeny of the transfected cell). Chemical methods are transfection techniques
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that use chemical carrier molecules to overcome the cell-membrane barrier. In these methods
cationic polymer, calcium phosphate, cationic lipid or cationic amino acid can be used.
Physical methods enable the direct transfer of nucleic acids into the cytoplasm or nucleus by
physical or mechanical means, e.g. by electroporation or nanoparticles [3].
In this work plasmid sequence of channelrhodopsin-2 is used to transfection evaluation.
Channerhodopsin is light activated protein isolated from Chlamydomonas reinhardtii, which
after activation, acts as divalent cation pump. Using this protein cell depolarization can be
controlled simply by illumination [4].
2. MATERIAL AND METHODS
HEK293 cells were cultivated at 37° and 5% CO2 in EMEM with 10% FBS, 1% P/S, 1% L-
Glutamine (SIAL) and passaged once a week up to thirty passages. Transfection was made at
9.2 mm2 cultivation dishes after 48 hour incubation. ChR2 is in this case marked by yellow
fluorescence probe for easy transfection efficiency tracking. Cell confluence at the confocal
petri dish between transfection was 50-70%.
In the chemical transfection we used polymer 1mg/ml polyethylenimine (PEI, Polysciences)
for transfection of ChR2 ion channel. PEI condenses DNA into positively charged particles
that bind to anionic cell surfaces or is endocytosed by the cells and the DNA released into the
cytoplasm [5]. Chemical transfection protocol is based on mixing DNA with PEI in solvent,
10 minutes incubation, adding fresh medium a replacing cultivation medium with transfection
mix.
In the physical transfection approach we used magnetic nanoparticles MATRA reagent (IBA)
which binds DNA and formed complex is using magnetic field transported into cytoplasm. In
this approach working protocol can be described as: solving DNA in serum free medium,
adding MATRA reagent, cultivation 20 minutes at RT, adding cultivation medium and
replacing old medium with transfection mix. In the next step cultivation plate is placed on
strong magnet for 5-20 minutes and culture is incubated O/N. Transfection reagent amounts
can be for both methods found in Table 1.
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Table 1: Transfection reagents
Surface
(sm2)
DNA (µg) Transfection
reagent (µl)
Solvent (µl) Cultivation
medium (ml)
9.2 2.59 8.00 (PEI)
110 (150 mM
NaCl) 1.94
9.2 2.59 2.91
(MATRA)
194 (EMEM) 1.94
Monitoring of transfection efficiency (fluorescence) was performed on the confocal laser
scanning microscope Leica TCS SP8 X equipped with the picosecond White Light Laser.
3. RESULTS AND DISCUSSION
As we can see on the figure 1. PEI mediated transfection efficiency is quite higher than
MATRA based transfection after the same time from transfection (only fluorescence cells
express ChR2). On the other hand, increasing the duration of the experiment to 48 hours
makes the method using MATRA reagent more successful, while the cell quality with PEI is
getting worse. The results of the experiment were evaluated by subjective view only.
Figure 1.: Results of experiment after 24 hours: PEI based transfection (left), MATRA based transfection (right)
4. CONCLUSION
Two transfection method of ChR2 is compared in this paper. By protocol optimization we
obtained results with good cell quality suitable for electrophoresis and quite high
fluorescence. For evaluation only subjective view is used but in the next work automatic
algorithm for efficiency evaluation will be developed.
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5. ACKNOWLEDGEMENT
The article was supported by grant project GACR P102/11/1068, European Regional
Development Fund - Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123) and by project
FEKT-S-14-2300 A new types of electronic circuits and sensors for specific applications.
6. REFERENCES
[1] Graham F. L., Smiley J., Russell W. C., and. Nairn R.: Journal of General Virology, 36 (1977), 59-72
[2] Thomas P. and Smart T. G.: Journal of Pharmacological and Toxicological Methods, 51 (2005), 187-200
[3] Kim T. K., and Eberwine J. H.: Analytical and Bioanalytical Chemistry, 397 (2010), 3173-3178.
[4] Nagel G., Szellas T., Huhn W., Kateriya S., Adeishvili N., Berthold P., Ollig D., Hegemann P., and
Bamberg E.: Proceedings of the National Academy of Sciences, 100 (2011), 13940-13945.
[5] Steitz B., Hofmann H., Kamau S. W., Hassa P. O., Hottiger M. O., Von Rechenberg B., Hofmann-
Amtenbrink M., and Petri-Fink A.: Journal of Magnetism and Magnetic Materials, 311 (2007), 300-305.
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CONTRIBUTIONS OF CYTOCHROMES P450 TO DETOXIFICATION
OF A HUMAN CARCINOGEN ARISTOLOCHIC ACID I IN HUMAN
AND RAT LIVERS
Marie STIBOROVA1*
, Frantisek BARTA1, Katerina LEVOVA
1, Petr HODEK
1, Eva FREI
2,
Heinz H. SCHMEISER3, Volker M. ARLT
4
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech
Republic
2 Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center
(DKFZ), In Neuenheimer Feld 280, 69 120 Heidelberg, Germany
3 Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), In
Neuenheimer Feld 280, 69120 Heidelberg, Germany
4 Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King’s
College London, London, United Kingdom
Abstract
Aristolochic acid (AA) causes a specific nephropathy, Aristolochic acid nephropathy, and
urothelial malignancies. The major component of AA, AAI, is predominant to be responsible
for these diseases. This carcinogen is detoxified by its O-demethylation to AAIa catalyzed by
cytochrome P450 (CYP) enzymes. Human CYP1A2, followed by CYP2C9, 3A4 and 1A1, are
major enzymes contributing to catalysis of this reaction in human liver. In rat liver, the
CYP2C and 1A enzymes are most efficient in AAI detoxification.
1. INTRODUCTION
Aristolochic acid (AA), a plant nephrotoxin and carcinogen, causes aristolochic acid
nephropathy (AAN) and its associated urothelial malignancy, and is hypothesized to be
responsible for Balkan endemic nephropathy (BEN). [1,2]. The major component of AA,
aristolochic acid I (AAI), is the predominant compound responsible for these diseases [2].
In contrast to the findings that AAI might directly cause interstitial nephropathy, metabolic
activation of AAI to species forming DNA adducts is a necessary step for AA-induced
malignant transformation [3-7]. Indeed, exposure to AA was demonstrated by the
identification of specific AA-DNA adducts in urothelial tissue of AAN and BEN patients [2-
4]. The most abundant DNA adduct detected in patients is 7-(deoxyadenosin-N6-yl)-
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aristolactam I (dA-AAI), which causes characteristic AT TA transversions. Such AT TA
mutations have been observed in the TP53 tumor suppressor gene in tumors from AAN and
BEN patients [3,4], indicating a probable molecular mechanism associated with AA-induced
carcinogenesis [2]. AA has been classified as a Group I carcinogen in humans by the
International Agency for Research on Cancer.
The concentration of AAI in organisms is crucial for both renal injury and induction of
malignant transformations initiated by activated AAI. Beside the ingested amounts of AAI,
metabolism of this compound dictates its effective concentration, thereby modulating also the
clinical consequences of exposure. A major metabolite of AAI formed under aerobic (i.e.
oxidative) conditions in vitro and in vivo is its O-demethylated product, AAIa. This
metabolite has been suggested to be a detoxication product of AAI [2-8]. One of the common
features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy
and cancer. We have recently suggested that beside differences in the cumulated dose of AAI
and the duration of AAI intake [6-8], differences in the activities of the enzymes catalyzing
the biotransformation (detoxication and/or activation) of AAI could be the reason for this
individual susceptibility. Hence, the identification of enzymes principally involved in the
metabolism (detoxication and/or activation) of AAI in humans and a detailed knowledge of
their catalytic specificities is of major importance.
Recent studies have indicated that human and rodent CYPs of the 1A subfamily are the major
enzymes oxidizing AAI to AAIa under aerobic (i.e. oxidative) conditions in vitro and in vivo
(for a review see [8]). In this study, we evaluated contribution of individual CYP enzymes
expressed in human liver to detoxification of AAI to AAIa and compared their contribution
with that of CYPs expressed in liver of rats.
2. MATERIAL AND METHODS
Microsomes isolated from insect cells transfected with baculovirus constructs containing
cDNA of human and rat CYPs and expressing POR (Supersomes ) and human and rat
hepatic microsomes were used as the enzyme systems oxidizing AAI. HPLC was used to
separate and identify AAI and its metabolites [9].
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3. RESULTS AND DISCUSSION
Using human and rat CYP enzymes recombinantly expressed in Supersomes , the enzymes
catalyzing oxidation of AAI to AAIa were indentified [9]. Human and rat CYPs of the 1A
subfamily are the major enzymes oxidizing AAI [9,10]. Other CYPs such as human and rat
CYPs of the 2C subfamily and human CYP3A (CYP3A4/5), 2D6, 2E1 and 1B1, also form
AAIa, but with more than one order of magnitude lower efficiency than CYP1A [9,10].
However, human/rat CYP1A1 and 1A2 orthologs exhibit species-species differences in AAI
preference and rates of its oxidation. Human CYP1A1 was found to be more effective to O-
demethylate AAI than human CYP1A2, whereas rat CYP1A2 oxidizes this compound more
efficiently than rat CYP1A1 [9-12].
Detoxification of AAI to AAIa was catalyzed also by human and rat hepatic microsomes.
These subcellular fractions of human and rat livers exhibited similar efficiencies to oxidize
AAI. Based on the data showing the velocities of AAI oxidation to AAIa by recombinant
CYPs and the expression levels of human CYP enzymes in human and rat hepatic
microsomes, contributions of individual CYP enzymes to AAI oxidation in human and rat
hepatic microsomes were estimated. The highest contribution to AAI oxidation in human
hepatic microsomes is attributed to CYP1A2 (~47.5%), followed by CYP2C9 (~15.8%),
CYP3A4 (~10.5%), and CY1A1 (~8.3%). Even though the activity of human recombinant
CYP1A1 to oxidize AAI is highest among all tested human CYPs, because of low expression
of this enzyme in human livers (<0.7%), its contribution to this reaction in human hepatic
microsomes is lower than that of CYP1A2, 2C9 and 3A4. In the case of rat hepatic
microsomes, the highest contribution to AAI oxidation to AAIa was attributed to CYPs of a
2C subfamily (~83.5%), followed by CYP1A (~17%). Other CYP enzymes expressed in
human and rat liver have essentially no AAI oxidation activity in human hepatic microsomes.
4. CONCLUSION
Using human and rat hepatic microsomes as well as human and rat recombinant CYP
enzymes, human CYP1A2, followed by CYP2C9, 3A4 and 1A1, were found to be the major
enzymes contributing to detoxification of AAI in human liver. In rat liver, the CYP2C and 1A
enzymes are most efficient in AAI detoxification.
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5. ACKNOWLEDGEMENT
Supported by GACR (grant 14-8344S) and Charles University (grant UNCE 204025/2012).
6. REFERENCES
[1] Debelle FD, Vanherweghem JL, Nortier JL: Kidney International, 74 (2008), 158-169
[2] Schmeiser HH, Stiborova M, Arlt VM: Current Opinion in Drug Discovery and Development, 12 (2009),
141-148
[3] Arlt VM, Stiborova M, vom Brocke J, et al.: Carcinogenesis 28 (2007), 2253-2261
[4] Grollman A.P., Shibutani S., Moriya M., et al.: Proceedings of American Chemical Society U.S.A., 104
(2007), 12129-12134
[5] Chen CH, Dickman KG, Moriya M, et al.: Proceedings of American Chemical Society U.S.A., 109 (2012),
8241-8246
[6] Stiborova M, Frei E, Arlt VM, et al.: Mutation Research, 658 (2008), 55-67
[7] Stiborová M, Frei E, Schmeiser HH: Kidney International, 73 (2008), 1209-1211
[8] Stiborová M, Martínek V, Frei E, et al.: Current Drug Metababolism 14 (2013), 695-705
[9] Stiborová M, Levová K, Bárta F, et al.: Toxicoogical. Sciences 125 (2012), 345-358
[10] Levová K, Moserová, M, Kotrbová V, et al.: Toxicoogical. Sciences 121 (2011), 43-56
XV. Workshop of Physical Chemists and Electrochemists
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OPTICAL AND SPECTROELECTROCHEMICAL STUDY OF
INTERACTION BETWEEN MESO-
TETRAKIS(4-SULPHONATOPHENYL)PORPHYRIN DERIVATIVES
AND CYCLODEXTRINS IN AQUEOUS SOLUTION
Juraj DIAN1*
, Jindřich JINDŘICH2, Jiří MOSINGER
3
1 Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in
Prague, Ke Karlovu 3, 121 16 Prague 2, Czech Republic
2 Department of Organic Chemistry, Faculty of Sciences, Charles University in Prague, Hlavova 2030, 128 40,
Prague 2, Czech Republic
3 Department of Inorganic Chemistry, Faculty of Sciences, Charles University in Prague, Hlavova 2030, 128
40, Prague 2, Czech Republic
Abstract
Optical and electrochemical properties of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin
(TPPS4) and its zinc complex (ZnTPPS4) and their interactions with various cyclodextrin
derivatives in aqueous solutions were studied. The interaction was monitored by UV/VIS
absorption detected both by UV/VIS spectrophotometer and in a optical thin layer
spectroelectrochemical cell at various potentials. The measurements revealed strong
interaction of the TPPS4 and ZnTPPS4 with cyclodextrins.
1. INTRODUCTION
Porphyrin photosensitizers TPPS4 and ZnTPPS4 like many other porphyrin derivatives
photosensitize production of singlet oxygen (1
g) whose effects on living cells are the basis of
photodynamic therapy (PDT) [1]. One of the main goals in PDT is the transport of a
photosensitizer to the tumor in aqueous environment. Cyclodextrins [2] (CDs) are cyclic
oligosaccharides that consist of various numbers of glucopyranose units. They form host-
guest complexes with many organic compounds and are well soluble in water. These two
properties are basis for the application of CDs as carriers of various hydrophobic
pharmaceutical compounds in aqueous solutions. In the present study we focused on the effect
of host-guest formation of TPPS4 and ZnTPPS4 with CDs in aqueous solutions. We studied
influence of CD concentration and applied external potential on absorption changes of TPPS4
and ZnTPPS4.
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2. MATERIAL AND METHODS
Native -cyclodextrin ( -CD, Sigma-Aldrich), 2-hydroxypropyl- -cyclodextrin (hp- -CD,
Aldrich) were used as received. TPPS4 and ZnTPPS4 were synthesized and purified as
described elsewhere [3]. UV-VIS absorption was measured on a Varian Carry IE
spectrophotometer using 10 mm quartz cuvettes. Spectroelectrochemical (SE) measurements
were performed using optical thin-layer electrochemical (OTTLE) cell by Hartl [4] with Pt
mesh electrode and Pt pseudoreference electrode. The optical path was ca 0.2 mm. Cyclic
voltammetry experiment was performed by Autolab PGSTAT101 potentiostat with NOVA
software, absorption experiment by Avantes AvaSpec ULS3648TEC optical fiber
spectrometer (resolution ~2 nm) with AvaLight DHc deuterium halogen lamp. Experiments
were performed in 0.02 M phosphate buffer (pH=7) at room temperature (20-22°C), solution
were bubbled for 10 minutes with argon prior to SE measurements.
3. RESULTS AND DISCUSSION
Absorption spectra of TPPS4 (Fig. 1A) and ZnTPPS4 in the presence of various amounts of
CDs revealed bathochromic shift of Soret band from 413 up to 420 nm and from 420 to 428,
respectively. The absorption changes can be easily observed in difference absorption spectra
(Fig. 1B) and can be attributed to the formation of a host-guest complex between porphyrin
and hp- -CD [1].
A B
Figure 1.: A. Structure of TPPS4, B. Difference absorption spectra of 1µM TPPS4 in the presence of 0, 5, 10, 20
and 100 µM hp- -CD in 0.02 M phosphate buffer (pH=7).
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Absorption spectra of TPPS4 and ZnTPPS4 in 0.1 M Na2SO4 at pH=7 measured by means of
an OTTLE cell are in Fig. 2A. Cyclic voltammograms of ZnTPPS4 in the presence of various
amounts of -CD (0,1 and 10 mM) are depicted in Fig. 2B. Evidently, host-guest interaction
stabilizes TPPS4 against electrochemical changes [4]. During CV scans absorption spectra
changes of ZnTPPS4 were recorded (not shown) at various potentials (relative to Pt pseudo-
reference electrode) and quasireversible changes in the intensity of Soret band were observed.
300 400 500 600 700 8000.00
0.05
0.10
0.15
0.20 ZnTPPS4
Ab
so
rba
nce
[O
D]
Wavelength [nm]
0.0
0.2
0.4
0.6
0.8 TPPS4
-1.0 -0.5 0.0 0.5 1.0
-0.2
-0.1
0.0
0.1 1 mM ZnTPPS4
1 mM ZnTPPS4 + 10 mM -CD
I [m
A]
U vs. Pt [V]
0.1 M Na2SO
4
0.02 M phosphate buffer pH=7
scan rate = 2 mV/s
A B
Figure 2.: A. Absorption spectra of 1mM TPPS4 and ZnTPPS4 aqueous solutions in 0.1 M Na2SO4 and 0.02 M
phosphate buffer pH=7 at V=0, B. Cyclic voltammograms of 1mM ZnTPPS4 without and in the presence of 10
mM -CD, scan rate 2 mV/s.
4. CONCLUSION
The presented study revealed strong interaction between TPPS4 and ZnTPPS4 porphyrins
with CDs. The interaction results in bathochromic shifts of the Soret band and stabilize the
studied porphyrins against electrochemical changes.
5. REFERENCES
[1] Mosinger J, Deumié M, Lang K, Kubát P, Wagnerová D. M.:J. Photochem. Photobiol., 130 (2000), 13-20
[2] Crini G.: Chem. Rev. 114 (2014), 10940–10975.
[3] Kubát P, Mosinger J.: J. Photochem. Photobiol. A, 96 (1996), 93-97
[4] Krejčik M, Daněk M, Hartl F.: J. Electroanal. Chem., 317 (1991), 179-187
[5] Kano K., Kitagishi H, Sone Y, Nakazawa N, Kodera M.: Eur. J. Inorg. Chem. (2006), 4043-4053
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VACUOLAR-ATPASE-MEDIATED INTRACELLULAR
SEQUESTRATION OF ELLIPTICINE CONTRIBUTES TO DRUG
RESISTANCE IN NEUROBLASTOMA CELLS
Jan HRABETA1, Tomas GROH
1,2, Mohamed Ashraf KHALIL
1, Jitka
POLJAKOVA2,Vojtech ADAM
3,4, Rene KIZEK
3,4, Jiri UHLIK
5, Helena DOKTOROVA
1,
Tereza CERNA2, Eva FREI
6, Marie STIBOROVA
2*, Tomas ECKSCHLAGER
1
1 Department of Pediatric Hematology and Oncology, 2
nd Medical School, Charles University and
University Hospital Motol, V Uvalu 84, 150 06 Prague 5, Czech Republic
2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague
2, Czech Republic
3 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno,
Zemedelska 1, 613 00 Brno, Czech Republic
4 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616
00 Brno, Czech Republic
5 Department of Histology and Embryology, 2
nd Medical School, Charles University, V Uvalu 84, 150
06 Prague 5, Czech Republic
6 Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research
Center (DKFZ), Im Neuenheimer Feld 280, 69 120 Heidelberg, Germany
Abstract
The up-regulation of a vacuolar (V)-ATPase gene is one of the factors associated with
development of resistance of UKF-NB-4 cells to ellipticine. It corresponds to the finding that
levels of V-ATPase protein expression are higher in the ellipticine-resistant UKF-NB-4ELLI
line than in the ellipticine-sensitive UKF-NB-4 cell line. Ellipticine induced cytoplasmic
vacuolization in these cells and is sequestrated in these vacuoles. A V-ATPase inhibitor
bafilomycin A and/or the lysosomotropic drug chloroquine enhanced the ellipticine-mediated
apoptosis, decreased ellipticine-resistance and formation of ellipticine-derived DNA adducts,
one of the most important DNA-damaging mechanisms responsible for ellipticine
cytotoxicity.
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1. INTRODUCTION
Neuroblastoma is a malignant tumor consisting of neural crest derived undifferentiated
neuroectodermal cells [1,2]. Unfortunately, little improvement in therapeutic options in high
risk neuroblastoma has been made in the last decade, requiring a need for the development of
new therapies.
Recently, we suggested a novel treatment for neuroblastomas, utilizing a drug targeting DNA,
the plant alkaloid ellipticine. We found that exposure of human neuroblastoma IMR-32, UKF-
NB-3 and UKF-NB-4 cell lines to this agent resulted in strong inhibition of cell growth,
followed by induction of apoptosis [3,4]. These effects were associated with formation of two
major covalent ellipticine-derived DNA adducts, identical to those formed by the cytochrome
P450 (CYP)- and peroxidase-mediated ellipticine metabolites, 13-hydroxy- and 12-
hydroxyellipticine [3,4]. Nevertheless, this drug is unfortunately capable of inducing
resistance in neuroblastoma cells. Ellipticine resistance in neuroblastoma is caused by a
combination of overexpression of Bcl-2, efflux or degradation of the drug, downregulation of
topoisomerases and the up-regulation of vacuolar (V)-ATPase [5]. The mechanism of V-
ATPase contribution to induction of resistance to ellipticine in the ellipticine-resistant UKF-
NB-4ELLI
cell line was investigated in this work.
2. MATERIAL AND METHODS
UKF-NB-4 and UKF-NB-4ELLI
cells were treated with ellipticine and analyzed for its
cytotoxicity by the MTS test and apoptosis development by Annexin V/DAPI labeling.
Formation of lysosomes and ellipticine sequestration was analyzed by fluorescence and
electron microscopy. The method of Western blot, employing antibodies against V-ATPase
(ATP6V0D1 membrane domain) protein, was utilized to evaluate expression of this protein.
The 32
P-postlabling method to detect and quantify BaP-derived DNA adducts [3,4].
3. RESULTS AND DISCUSSION
Exposure to ellipticine induced apoptosis in human neuroblastoma UKF-NB-4 cells sensitive
to ellipticine and in the cells resistant to this drug (UKF-NB-4ELLI
) and inhibited their growth.
The up-regulation of a vacuolar (V)-ATPase gene is one of the factors associated with
resistance development [5]. In accordance with this finding, we found in this study that levels
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of V-ATPase protein expression are higher in the ellipticine-resistant UKF-NB-4ELLI
line than
in the parental ellipticine-sensitive UKF-NB-4 cell line.
Treatment of ellipticine-sensitive UKF-NB-4 and ellipticine-resistant UKF-NB-4ELLI
cells
with ellipticine induced cytoplasmic vacuolization and ellipticine is concentrated in these
vacuoles. Confocal microscopy and staining of the cells with a lysosomal marker suggested
these vacuoles as lysosomes. Transmission electron microscopy and no effect of an autophagy
inhibitor wortmannin ruled out autophagy. Pretreatment with a V-ATPase inhibitor
bafilomycin A and/or the lysosomotropic drug chloroquine prior to ellipticine enhanced the
ellipticine-mediated apoptosis and decreased ellipticine-resistance in UKF-NB-4ELLI
cells.
Moreover, pretreatment with these inhibitors increased formation of ellipticine-derived DNA
adducts, one of the most important DNA-damaging mechanisms responsible for ellipticine
cytotoxicity. Therefore, we can postulate that resistance to ellipticine in the tested
neuroblastoma cells is associated with V-ATPase-mediated vacuolar trapping of this drug,
which may be decreased by bafilomycin A and/or chloroquine.
4. CONCLUSION
Based on these results, we can conclude that the decrease in ellipticine-mediated cytotoxicity
on UKF-NB-4 cells as well as in induction of resistance to ellipticine in the ellipticine-
resistant UKF-NB-4ELLI
cell line is associated with vacuolar trapping of this drug, which may
be abolished by bafilomycin A or by chloroquine. Therefore, therapeutic implications could
be derived from this study. In principle, the components of the endocytic/lysosomal pathway
could be molecular targets for a combination therapy of neuroblastoma with chemotherapeutic
drugs and probably also for that of other cancers.
5. ACKNOWLEDGEMENT
The work has been supported by GACR (grants P301/10/0356 and 14-8344S) and Charles
University (grant UNCE 204025/2012).
6. REFERENCES
[1] Schwab M, Westermann F, Hero B, et al.: Lancet Oncol., 4 (2003), 472-480.
[2] Brodeur G.M.: Nat Rev Cancer, 3 (2003), 203–216.
[3] Poljaková J, Eckschlager T, Hrabeta J et al.: Biochem Pharmacol, 77 (2009), 1466–1479.
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[4] Stiborová M, Frei E: Curr Med Chem, 21 (2014), 575-591.
[5] Procházka P, Libra A, Zemanová Z et al:. Cancer Sci 103 (2012), 334–341.
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ROLE OF ZINC IONS IN ADVANCED PROSTATE CANCER MODEL
Jaromír GUMULEC1,2*
, Markéta SZTALMACHOVÁ1,2
, Jan BALVAN1,2
, Michal
MASAŘÍK1,2
1 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00,
Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
Advanced prostate cancer is difficult-to-treat disease with unsatisfactory survival statistics.
With this regard, numerous therapeutic approaches have developed over the years. Apart from
surgery, targeting testosterone-related pathways brought some improvements. Nevertheless,
advanced cancer of prostate is often characterized by resistance to hormonal therapy due
development of specific clones of cancer cells. Accordingly, the sensitivity of advanced
prostate cancer to commonly used cytostatics is also decreased. Thus, an advanced prostate
cancer model cell line PC-3 was adopted in our lab for in depth analysis. Interestingly,
dramatically high cytotoxic effect of zinc ions in this model was determined using
conventional MTT and real-time RTCA assays. In the next step, a zinc-resistant advanced
prostate cancer model was created using natural selection and this cell line was profiled for
gene expression patterns. NFKB, BAX, BCL, and Metallothionein were significantly
associated with the exposure of zinc sulphate treatment. This indicate and confirm the well-
established connection of zinc with important cellular processes such as the regulation of
apoptosis, metabolism, metal-buffering and oxidative stress. In the next step the content of
various fractions of zinc – free zinc and bound zinc fraction – was detected before and after
the exposure of zinc treatment on this prostate cancer model in both medium and in cells.
Based on this approach a novel therapeutic strategy of advanced prostate cancer based on the
modulation of intra-tumor zinc levels may be developed.
Acknowledgement
The financial support from CEITEC CZ.1.05/1.1.00/02 is greatly acknowledged.
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THE INFLUENCE OF VERTEX POTENTIAL AND MULTIPLE SCAN
VOLTAMMETRY ON THE FORMATION OF 8-OXOGUANINE
Libor GURECKY1, Iveta PILAROVA
1, Libuse TRNKOVA
1,2*
1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech
Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00
Brno, Czech Republic
Abstract
The electrochemical behavior of guanosine (Guo) and guanosine-5´-monophosphate (GMP) at
pencil graphite electrode (PeGE) in the phosphate-acetic buffer is examined by cyclic
voltammetry (CV). The oxidation peaks of both compounds appear at 1.1 V naturally, but
using a multiple cycle we observed an additional peak. It was found that this peak
corresponds to oxidation of 8-oxo-7,8-dihydroguanine (8-oG). To determine the potential of
8-oG formation we changed positive vertex potentials in the range from 0.9 to 1.4 V vs.
Ag/AgCl/3MKCl reference electrode. The lowest vertex potential at which the 8-oG provided
response corresponds to 1.3V for both Guo and GMP (pH 4.16). The most important is the
fact that the appearance of the 8-oG peak always occurs after the first cycle of CV.
1. INTRODUCTION
Purine derivatives are very important in biological processes due to the fact that cytosine and
guanine act as monomer units of nucleic acids. This study deals specifically with the
guanine`s corresponding nucleoside guanosine and GMP. Guanosine is formed through a two-
step mechanism with 8-oxoguanine as intermediate and involves the total loss of four
electrons and four protons [1,2]. Recent studies show that 8-oG can be used as a predictor for
individual radiosensitivity. Urinary 8-oG can be attributed entirely to DNA repair, mainly to
OGG1 (8-oxo7,8-dihydroguanine glycosylase) [3]. GMP, as one of the four main ribosyl
nucleotides in RNA, can be synthesized in the human body. It plays a key role in many
functions in cellular metabolism and cardiac activities such as gastrointestinal tract repair,
influencing the metabolism of fatty acids, enhancing imine response, etc. [4]. Several
procedures for computer processing of the CV measurements were described. The elimination
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voltammetric procedure (EVP), eliminating some chosen particular currents from
voltammetric measurement and preserving others to enhance signals or show some of signals
which can be hidden in regular voltammetry, was used [5].
2. MATERIALS AND METHODS
Chemicals
Guanosine (Guo) and guanosine-5´-monophosphate (GMP) were purchased from Sigma
Aldrich (St. Louis, USA). The concentration was 10 µM and was determined by UV
spectroscopy. The solutions were added into phosphate-acetate buffer (supporting electrolyte;
pH 4.16) with adjusted ionic strength I = 0.18 M (NaCl).
Cyclic voltammetry (CV)
Voltammetric experiments were performed using the electrochemical analyzer AUTOLAB
PGSTAT 30 (Ecochemie, Utrecht, The Netherlands) in connection with NOVA software.
Electrochemical measurements were carried out in a free-electrode system. PeGE (Tombow
0.5 HB, Japan) with an effective area of 16 cm2 was used as working electrode. Ag/AgCl/3M
KCl was used as a reference electrode and platinum wire as an auxiliary electrode. The
experimental conditions were as follows: time of adsorption 5 s, scan rate 200 mV/s; 400
mV/s and 800 mV/s, and potential range from 0 V to upper vertex potential (0.9 V; 1.0 V; 1.1
V; 1.2 V; 1.3 V; 1.4 V). The results from CV are used for EVP.
3. RESULTS AND DISCUSSION
We studied the dependence of changing the upper vertex potential in CV with 5 scans in the
oxidation process for Guo and GMP on the PeGE. The oxidation signal for GMP and Guo
appeared at 1.1 V. The second signal appeared at 0.55 V after the first cycle and refers to 8-
oG. We evaluated this second signal using the EVP with the following equation:
f(I) =17.485I – 11.657 I1/2 – 5.8284 I2
where I is the reference scan rate (400mV/s), I1/2 and I2 are half and double of reference scan
rate, respectively. This elimination function f(I), named E4, eliminates the charging and
kinetic current components and conserves the difussion current component. With more
negative vertex potential than 0.9 V no signals were observed in all LSV and EVP curves. The
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8-oG signal appeared in all cycles but not in the first one. The difference between Guo and
GMP was observed with increasing vertex potential at higher potentials than 1.1 V.
Figure 1: CV of the 1st and 2nd cycle of Guo with different upper vertex potentials. Inset: detail view showing
oxidation peak of 8-oG.
Figure 2: CV of the 1st and 2nd scan of GMP with different upper vertex potentials inset: oxidation of 8-oG
4. CONCLUSION
Using the CV technique we investigated the oxidation processes for Guo and GMP in
dependence on the upper vertex potential. After the first scan, a second peak appeared
corresponding to 8-oG. No peak was observed using only one scan even though we performed
adsorption on the electrode. By changing the upper vertex potential to 1.0 V, the peak
-1.00E-06
4.00E-06
9.00E-06
1.40E-05
1.90E-05
2.40E-05
2.90E-05
0.4 0.6 0.8 1 1.2
I/A
E/V
Oxidation of guanosine in the dependence on upper vertex
potential (scan rate 400 mV/s; pH 4.16; c = 10 µM)
0.9 V 1.scan
0.9 V 2.scan
1.0 V 1.scan
1.0 V 2.scan
1.1 V 1.scan
1.1 V 2.scan
1.2 V 1.scan
1.2 V 2.scan
1.3 V 1.scan
1.3 V 2.scan
0.00E+00
1.00E-06
2.00E-06
3.00E-06
4.00E-06
0.4 0.6
I/A
E/V
8-oG
-3.00E-06
7.00E-06
1.70E-05
2.70E-05
3.70E-05
4.70E-05
0.4 0.6 0.8 1 1.2 1.4
I/A
E/V
Oxidation of GMP in the dependence on upper vertex potential
(scan rate 400 mV/s; pH 4.16; c = 10 µM)
1.0 V 1.scan
1.0 V 2.scan
1.1 V 1.scan
1.1 V 2.scan
1.2 V 1.scan
1.2 V 2.scan
1.3 V 1.scan
1.3 V 2.scan
1.4 V 1.scan
1.4 V 2.scan
0.00E+00
5.00E-07
1.00E-06
1.50E-06
2.00E-06
0.4 0.6
I/A
E/V
8-oG
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disappeared for both compounds. The lowest upper vertex potential limit for the detection of
8-oG for GMP was 1.1 V while 1.2 V was the threshold vertex potential in the case of Guo.
5. ACKNOWLEDGEMENT
This research was supported by the following projects: (a) SIX CZ.1.05/2.1.00/03.0072 (b)
MUNI/A/1452/2014 (c) KONTAKT II LH 13053 of the Ministry of Education, Youth and
Sports of the Czech Republic.
6. REFERENCES
[1] Brett A.M.O., Matysik F.M.: Bioelectrochemistry and Bioenergetics, 42 (1997), 111-116.
[2] Chen S.M., Wang C.H.: Bioelectrochemistry, 70 (2007), 2, 452-461.
[3] Roszkowski K., Olinski R.: Cancer Epidemiology Biomarkers and Prevention, 21 (2012), 629-634.
[4] Sun W., Xu L., Liu J. et al.: Croatia Chemica Acta, 86 (2013), 129-135.
[5] Trnkova L., Adam V., Kizek R. (Eds): Utilizing of Bio-electrochemical and Mathematical Methods in
Biological Research, Research Signpost, Kerala, India, Ch. 4 (2007), 51.
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VOLTAMMETRIC DETECTION OF DNA DAMAGE CAUSED BY
2-AMINOFLUORENE AND ITS METABOLITE
2-ACETYLAMINOFLUORENE
Andrea HÁJKOVÁ1*
, Jiří BAREK1, Vlastimil VYSKOČIL
1
1 Charles University in Prague, Faculty of Science, University Research Centre UNCE "Supramolecular
Chemistry", Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry,
Hlavova 2030/8, 128 43 Prague 2, Czech Republic
Abstract
The voltammetric investigation of the interaction between selected derivatives of fluorene and
double-stranded DNA (dsDNA) was conducted to characterize their damaging effects on the
dsDNA structure. The interaction was investigated firstly by differential pulse voltammetry
(DPV) at a bare glassy carbon electrode (GCE), when dsDNA and 2-aminofluorene (2-AF) or
its metabolite 2-acetylaminofluorene (2-AAF) were present in the measured solution. Afterwards,
square-wave voltammetry (SWV) was performed at a DNA biosensor (prepared by adsorption
of dsDNA onto the GCE surface (dsDNA/GCE)), after its incubation in the solution of 2-AF or
2-AAF for various times and at various concentrations of 2-AF or 2-AAF. The intercalation of
the studied compounds between the dsDNA base pairs was observed by both detection methods.
1. INTRODUCTION
Detection of specific mutations in DNA sequences and studies of supramolecular interactions
of DNA with various dangerous organic compounds are one of the most important research
areas of bioanalytical chemistry [1,2]. Serious diseases, such as cancer, can be caused by
relatively small changes in the DNA structure [3]. Many derivatives of fluorene, such as 2-AF
and 2-AAF, are chemical carcinogens and mutagens. In the past, their interaction with DNA
formed the basis of genetic toxicity testing [4]. In this work, highly sensitive voltammetric
techniques for monitoring DNA damage were used [2,5] to characterize detrimental effects of
2-AF and 2-AAF.
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2. MATERIAL AND METHODS
Stock solutions of 2-AF (98%, Sigma-Aldrich, USA) and 2-AAF (> 98%, Sigma-Aldrich,
USA) were prepared in methanol. Different concentrations of low molecular weight salmon
sperm dsDNA (Sigma-Aldrich, USA) were prepared by its dissolving in a 0.1 mol L−1
phosphate
buffer of pH 6.7 (PB). Voltammetric measurements were carried out in a three-electrode
system – a platinum wire auxiliary electrode, a silver/silver chloride reference electrode
(3 mol L−1
KCl), and a GCE or a dsDNA/GCE as a working electrode.
3. RESULTS AND DISCUSSION
Two detection modes were used, both performed in the positive potential region: (i) DPV at
the bare GCE when both dsDNA and 2-AF or 2-AAF were present in the measured solution
and (ii) SWV at the dsDNA/GCE after its incubation in the solutions of 2-AF or 2-AAF.
Investigation of 2-AF or 2-AAF with dsDNA present in solution
Firstly, DPV at the GCE was performed in the solutions containing 2-AF (or 2-AAF) and
various concentrations of dsDNA. Various amounts of dsDNA were added to the 10.0 mL
solution of 1×10–4
mol L–1
2-AF (or 2-AAF) in the PB. The height of the 2-AF peak
decreased with the increasing concentration of dsDNA, and the peak potential was shifted
towards more positive potentials. This behavior can be attributed to the formation of the
intercalation 2-AF–dsDNA complex, since 2-AF present in the complex is more difficult to be
oxidized than the free form of 2-AF. In the case of 2-AAF, the height of its peak decreased
only when small amounts of dsDNA were added, and the peak potential was shifted towards
more positive potentials, too. However, when higher amounts of dsDNA were added, the
height of the 2-AAF peak increased, since it was affected by interfering signal from guanosine
oxidation, as potentials of 2-AAF and guanosine peaks are very similar and their peaks are
thus not well separated. Then, DPV at the GCE was performed in the solution of dsDNA
(γ = 1 mg mL–1
) in the PB, with various additions of 1×10–4
mol L–1
2-AF (or 2-AAF) into the
10.0 mL solution of dsDNA. The peak potentials of guanosine and adenosine units present in
the dsDNA structure were shifted towards more positive potentials and their height decreased
with the increasing concentration of 2-AF (or 2-AAF).
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Investigation of 2-AF or 2-AAF with the dsDNA/GCE biosensor
The electrochemical DNA biosensor was prepared by adsorption of salmon sperm dsDNA on
the polished (using the aluminum oxide suspense) GCE. For its preparation, optimum parameters
of the dsDNA adsorption were searched (a concentration of dsDNA, an adsorption potential,
and an adsorption time). The prepared dsDNA/GCE biosensor was used to monitor DNA
damage induced in the presence of the studied compounds. The biosensor was incubated for
various times and at various concentrations of the analyte in solution. SWV was carried out at
the biosensor in the pure PB to monitor the changes in the intensity of the oxidation signals of
guanine and adenine moieties before and after the interaction with 2-AF (or 2-AAF). It was
found that 2-AF exhibited both incubation time-dependent and concentration-dependent
damaging effects on dsDNA, causing a decrease of the guanosine and adenosine SWV peak,
while the peak of 2-AF, which was present at the electrode surface in the form of the 2-AF–
dsDNA complex, increased. The peaks of 2-AAF and guanosine were not separated, therefore,
only SWV peak of adenosine can be monitored, which decreased with the increasing
concentration of 2-AAF.
4. CONCLUSION
The interaction between the selected genotoxic derivatives of fluorene (2-AF and its metabolite
2-AAF) and dsDNA was investigated in this work by DPV at the bare GCE and by SWV at
the dsDNA/GCE. It was confirmed by both voltammetric techniques that 2-AF, as well as 2-AAF,
interacts with dsDNA, which affects the electrochemical signals of guanosine and adenosine
units present in the dsDNA structure. The predominant damaging interaction observed was
the intercalation of 2-AF and 2-AAF between the dsDNA base pairs, causing structural
changes and consecutive formation of double-strand breaks.
5. ACKNOWLEDGEMENT
This research was carried out in the framework of the Specific University Research (SVV
2015). A.H. thanks the Grant Agency of the Charles University in Prague (Project GAUK
430214/2014/B-CH/PrF), and J.B. and V.V. thank the Grant Agency of the Czech Republic
(Project P206/12/G151) for the financial support.
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6. REFERENCES
[1] Vyskocil V, Blaskova M, Hajkova A, et al.: Sensing in Electroanalysis, 7, University Press Centre, Pardubice
(2012), 141-162
[2] Hajkova A, Barek J, Vyskocil V: Electroanalysis, 27 (2015), 1, 101-110
[3] Vyskocil V, Labuda J, Barek J: Analytical and Bioanalytical Chemistry, 397 (2010), 1, 233-241
[4] Heflich RH, Neft RE: Mutation Research-Reviews in Genetic Toxicology, 318 (1994), 2, 73-174
[5] Labuda J, Vyskocil V: Encyclopedia of Applied Electrochemistry, Springer, New York (2014), 346-350
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MULTI-WALLED CARBON NANOTUBES AND THEIR DOUBLE-
STEP FUNCTIONALIZATION WITH ETOPOSIDE AND ANTISENSE
PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES
Zbynek HEGER1,2
, Amitava MOULICK1,2
, Hoai Viet NGUYEN1,2
, Monika
KREMPLOVA1,2
, Pavel KOPEL1,2
, David HYNEK1,2
, Ondrej ZITKA1,2
, Vojtech ADAM1,2
,
Rene KIZEK1,2*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
Herein we present the electrochemical characterization of binding capability of acidic
oxidized multi-walled carbon nanotubes (oMWCNTs) modified with poly(ethylene glycol)
towards common cytostatic drug etoposide. To obtain a multifunctional nanotransporter we
employed the phosphorothioate oligodeoxynucleotide (PODN), which could further extend
the possible biological effects of MWCNT-PEG-Etoposide complex.
1. INTRODUCTION
Over the past decade various approaches of MWCNTs functionalization have been exploited
to develop a multifunctional carbon-based platform for nanomedicinal applications. Acidic
oxidation of MWCNTs with nitric acid generates covalently bound functional groups [1]. One
of the most popular modifications of MWCNTs is tethering with biofunctional spacer -
poly(ethylene glycol) (PEG), particularly due to its biocompatibility and capability to extend
MWCNT modification possibilities and their blood circulation time. Moreover, MWCNT-
PEG offers a possibility of multi-functionalization [3], which is important for development on
innovative nanomaterials for next-generation theranostic nanomedicine. Etoposide (or VP-16)
is commonly used for the treatment of a variety of malignancies. In combination with suitable
antisense oligonucleotides, which are helpful tools for the in vivo regulation of gene
expression, etoposide offers powerful weapon to fight cancer. Thus, the present study aims on
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preparation and characterization of acidic oxidized MWCNT and their two-step modification
with etoposide and phosphorothioate oligodeoxynucleotides.
2. MATERIAL AND METHODS
Preparation of multiwall carbon nanotubes and PEGylation
2 mg of MWCNTs (Sigma Aldrich, St. Louis, MO, USA) was taken in an Eppendorf tube and
subsequently 1 mL of 68% HNO3 (Sigma Aldrich, MO, USA) in aqueous solution (w/w) was
added for its oxidation. The mixture was heated using a thermo-mixer (Eppendorf,
Hamburg, Germany) for 1 h at 80°C and 800 rpm. The sample was sonicated using an
ultrasonic bath (Bandelin, Berlin, Germany) for 15 min and centrifuged at 25 000 rpm at 20°C
for 10 min using a table top centrifuge machine (Eppendorf, Hamburg, Germany). The
supernatant was discarded and the MWCNTs were washed 6-7 times by centrifugation (25
000 rpm at 20°C for 10 min) with MiliQ water until the pH became 7.
To prepare PEGylated MWCNT 1 mL of crude PEG solution (40%, w/w) was mixed with 1
mL of MWCNT and resulting mixture was 20 min at 25oC. The solution was further
centrifuged (25 000 rpm at 20°C for 10 min) and the supernatant was discarded to remove
unbound PEG. The PEGylated MWCNT was re-dissolved in 1 mL of H2O. Purified
MWCNTs-PEG solution was stored at 4oC.
Differential pulse voltammetry
Differential pulse voltammetry (DPV) for detection of etoposide and the complexes of
etoposide with MWCNT and MWCNT-PEG was performed by using glassy carbon electrode
(GCE). Parameters for DPV analysis were initial potential 0 V; end potential 1 V; modulation
amplitude 0.1 V; modulation time 0.004s; interval time 0.1 s.
Square wave voltammetry
MWCNT-PEG-Eto complex was incubated with solution of PODNs (0; 1; 3; 5; 10 μM) and
after decantation; supernatant was removed and utilized for measurements by square wave
voltammetry (Metrohm, Herissau, Switzerland), using a standard cell with three electrodes.
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3. RESULTS AND DISCUSSION
MWCNTs are usually functionalized by harsh oxidative processes, such as refluxing in
concentrated acids, generating defects, which can serve as anchor moieties for further
modification [4]. Firstly, the interaction between oMWCNTs and etoposide was analyzed
using three different batches of complexes. The DPV results revealed that in case of the
highest applied concentration of etoposide (15 mM), the determined peak height was about
162.4 µA (corresponding to 46.4% recovery). Using lower applied concentration (12 mM),
relatively similar result (153.2 µA, recovery 54.8%) was obtained, which points at probable
saturation plateau of MWCNT-PEG complex. Interestingly, in the lowest applied
concentrations, higher recoveries were observed. This phenomenon is connected with
relatively short incubation time (1 h) and it could be expected that longer time will led to fully
saturation of MWCNT-PEG with etoposide. For identification of binding ability of PODNs to
MWCNT-PEG-Eto, square wave voltammetric technique was employed. As a results,
molecule of DNA exhibits typical CA peak (potential about -1.39 V). Fig. 1A shows the
calibration curve of employed PODN (sequence analog of Oblimersen). Fig. 1B presents the
PODNs concentrations (red bar), determined in discarded supernatant after incubation. It is
obvious that application of 1 and 3 μM PODNs led to maximum saturation of MWCNT-PEG-
Eto, thus no CA peak was identified in supernatant. In case of higher applied concentrations -
5 and 10 μM, PODNs presence in supernatant was observed (0.47 μM or 5.50 μM,
respectively).
Figure 1.: Loading efficiency of MWCNT-PEG-Eto. (A) Calibration curve of PODNs (0.08 - 20.00 μM) with
inset with corresponding square wave voltammograms. (B) Residual PODNs in supernatant after forming a
complex with MWCNT-PEG-Eto and subsequent decantation.
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4. CONCLUSION
Etoposide/PODNs loaded PEGylated MWCNTs were prepared with exceptional drug and
antisense nucleic acid loading capacities. The complex can be prepared by simple multi-step
process. However further biological studies have to be carried out to show the effects on
proliferation, target protein expressions of cancer cells and determine the complex behavior in
circulatory system.
5. ACKNOWLEDGEMENT
The work has been supported by League against cancer Prague (project 18257/2014-981).
6. REFERENCES
[1] Wang Z W, Shirley M D, et al.: Carbon, 47 (2009), 1, 73-79
[2] Hande K R.: European Journal of Cancer, 34 (1998), 10, 1514-1521
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EVALUATION OF ANTI-PAIIL IMMUNOGLOBULIN EFFICACY BY
MONITORING OF LUMINESCENT PSEUDOMONAS AERUGINOSA
Petr HODEK1*
, Lucie VAŠKOVÁ
1, Libuše NOSKOVÁ
1, Barbora BLÁHOVÁ
1, Michaela
WIMMEROVÁ2, Božena KUBÍČKOVÁ
1, Marie STIBOROVÁ
1
1 Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 40 Prague 2,
Czech Republic
2 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech
Republic
Abstract
Using a regular (ST 1763) and luminescent (lux) strains of Pseudomonas aeruginosa (PA) the
chicken yolk antibody prophylaxis against adhesion of this bacterium on epithelium cell lines
derived from normal or cystic fibrosis (CF) human lungs was examined. Antibodies (IgYs) of
two different chickens prepared against PA lectin, PAIIL, almost equally prevented PA
bacteria adhesion in both cell lines. In accordance with clinical data our results showed higher
susceptibility of CF cells to PA binding compared to a normal airway epithelium. Depending
on the PA handling (staining with fluorescent dye) and the way of PA detection
(fluorescence/luminescence) the results of IgY protection of cells differed accordingly. This
finding may suggest the interplay of various PA adhesion factors, which are or not affected by
bacteria treatment in the assay.
1. INTRODUCTION
The genetic disease called Cystic fibrosis (CF) is a disorder caused by mutations of the gene
coding for the CF transmembrane conductance regulator (CFTR) protein. Because of changes
in the lung CF patients are susceptible towards airway microbial infections with pathogens
such as Pseudomonas aeruginosa (PA). These infections usually turn to be chronic
endobronchial colonization, which makes the cystic fibrosis to be one of the most common
life-shortening disorders. The treatment of bacterial infections with antibiotics is frequently
ineffective because of the bacteria resistance or biofilm formation. To prevent the morbidity
and mortality of CF patients from bacterial infections there is a critical need to find new
effective therapies. While the CF gene therapy and corrections of CFTR function are studied,
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the CF patient immunization against pathogens is being examined, too. The immunization of
CF patients against bacterium virulence factors seems to be limited with impaired secretion of
protective immunoglobulins (IgG) in CF airways. Moreover, even when specific IgG binds
the pathogen bacteria inflammatory reactions are initiated. This process causes detrimental
changes of lung epithelium. Apparently, there is a need for so called “passive immunization”
via administered immunoglobulins, which do not trigger the inflammation. The chicken yolk
antibodies (IgYs) are well suited for this purpose since they, in contrast to mammalian IgGs,
do not induce inflammatory reaction when antigen is bound. Moreover, IgYs could be easily
prepared in large quantities (100 mg IgY/yolk). These properties make chicken yolk
antibodies to be an excellent tool for prophylaxis of bacterial infections.
2. MATERIAL AND METHODS
Preparation of antibody
Antibodies were prepared from egg yolks laid by chickens immunized with recombinant PA
lectin, PAIIL, as described elsewhere [1]. Pre-immune IgY sample (control) was purified
from eggs collected a week prior to the immunization. The presence of anti-PAIIL IgY was
determined on ELISA and Western blots using PAIIL and PA lysate as antigens, respectively.
Assay of bacterial adhesion on epithelial cells
The assay was performed according to Noskova et al. [2]. Immortalized epithelium cell lines
derived from normal (NuLi) or CF (CuFi) human lungs (ATCC) were stained with a
fluorescent dye PKH67, seeded onto well plates (24 wells) and incubated for 24 h at 37°C,
5% CO2 to form a confluent layer. A regular P. aeruginosa strain (ST 1763) or a
bioluminescent PAO1 (ST 549) containing a luxCDABE cassette (generous gift of Dr. Robert
E. W. Hancock, University of British Columbia, Vancouver, Canada) were labeled with a
fluorescent dye PKH26 or used as plain. The bacterial suspension with anti-PAIIL or control
IgYs (1 mg/ml), or PBS, was applied onto the well plates. After 2 hrs incubation non-adhered
bacteria were removed by extensive washing with PBS. The adhered PA cells on epithelial
cells were quantified (Ex 522 nm, Em 569 nm for PA; Ex 470 nm, Em 505 nm for
NuLi/CuFi, or measuring the PA luminescence) using spectrofluorometer (Tecan Infinite
M200 Pro). Results were expressed as a relative fluorescence ratio PA/NuLi or PA/CuFi as
well as the PA luminescence/epithelium cell fluorescence.
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3. RESULTS AND DISCUSSION
The adherence of bacteria to epithelial cells is an essential process in PA pathogenesis. The
bacterium possesses several specific adhesins, which significantly contribute to its virulence.
It is thought that airway surfaces of CF patients are lacking the sialylation of glycoconjugates
such as GM1, which enables the binding of PA via saccharide specific lectins, e.g. PAIIL [3].
To prevent the bacteria adhesion on host cells chicken yolk antibodies against the
recombinant PAIIL were prepared and tested. Their prophylactic properties against the PA
colonization of lung epithelial cells derived from normal (NuLi) or CF-patient (CuFi) lung
tissues has been already proven using an adhesion assay based on a dual fluorescence
determination of PA and epithelial cells [2]. To examine the efficacy of anti-PAIIL IgY
further, antibodies of two different chickens as well as another PA strain, a bioluminescent
PA-lux containing a luxCDABE cassette, were involved in the present study. Both
preparations of anti-PAIIL antibody almost equally prevented PA bacteria adhesion in used
cell lines. In accordance with clinical data our results showed higher susceptibility of CF cells
to PA binding compared to a normal airway epithelium. Interestingly, when in the adhesion
assay a regular or bioluminescent PA-lux strain is used, a different IgY protection of epithelial
cells was determined. These differences may reflect variances of the PA strains or most likely
the consequences associated with the PA handling during the assay. While the regular PA
strain is stained with a fluorescent dye, which consists in repeated sedimentation and re-
suspendation, the bioluminescent PA-lux strain is used as it is - without any manipulations.
One may expect that the fragile virulence factors such as flagella and fimbriae are partially
lost in the course of bacteria staining. Under such conditions other virulence factors, which
are tightly bound with the bacteria (e.g. lectins), would prevail in the bacteria adherence. This
phenomenon may explain the observed differences in the adhesion tests.
4. CONCLUSION
Chicken yolk antibodies against P. aeruginosa lectin PAIIL repeatedly proved their ability to
reduce PA bacteria adhesion on human airway epithelia cells under experimental condition
used.
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5. ACKNOWLEDGEMENT
The work has been supported by GAUK 1584814 and UNCE 204025/2012.
6. REFERENCES
[1] Hodek P, Trefil P, Šimůnek J, et al.: International Journal of Electrochemical Science, 5 (2013), 113-124
[2] Nosková L, Kubíčková B, Vašková L, et al.: Sensors (Basel), 16 (2015), 1945-1953
[3] Bryan R, Kube D, Perez A, et al.: American Journal of Respiratory Cell and Molecular Biology, 19 (1998),
269-277
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BENZO[A]PYRENE IS OXIDIZED BY RAT HEPATIC MICROSOMES
BOTH IN THE PRESENCE OF NADPH AND NADH
Radek INDRA1, Michaela MOSEROVÁ
1, Miroslav ŠULC
1, Volker M. ARLT
2, Marie
STIBOROVÁ1*
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech
Republic
2 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health,
King’s College London, United Kingdom
Abstract
Metabolism of benzo[a]pyrene (BaP) by hepatic microsomes of control (uninduced) rats and
by microsomes isolated from livers of rats treated with inducers of individual cytochrome
P450 (CYP) enzymes metabolize BaP in the presence of both a coenzyme of NADPH:CYP
reductase, NADPH, and a coenzyme of NADH:cytochrome b5 reductase, NADH. The results
indicate that NADH in these microsomal systems can act as a sole electron donor both for the
first and second reduction of CYPs in their reaction cycle.
1. INTRODUCTION
Benzo[a]pyrene (BaP) has been classified as human carcinogen (Group 1) by the International
Agency for Research on Cancer [1]. This is genotoxic carcinogen that covalently binds to
DNA after metabolic activation by cytochrome P450 (CYP) [2]. CYP1A1 and 1B1 were
found to be the most important enzymes in BaP bioactivation [2,3], in combination with
microsomal epoxide hydrolase (mEH). First, CYP1A1 oxidizes BaP to an epoxide that is then
converted to a dihydrodiol by mEH (i.e. BaP-7,8-dihydrodiol); then further bio-activation by
CYP1A1 leads to the ultimately reactive species, BaP-7,8-dihydrodiol-9,10-epoxide (BPDE)
that can react with DNA, forming adducts preferentially the 10-(deoxyguanosin-N2-yl)-7,8,9-
trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene adduct (dG-N2-BPDE adduct) in vitro and in
vivo [4-6]. BaP is, however, oxidized also to other metabolites, such as the other dihydrodiols,
BaP-diones and hydroxylated metabolites that are mainly the detoxification products. Even
though most of these metabolites are the detoxification products, BaP-9-ol is a precursor of 9-
hydroxy-BaP-4,5-epoxide that can form another adduct with deoxyguanosine in DNA [5-7].
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The CYP enzyme is a component of mixed-function-oxidase (MFO) system located in the
membrane of endoplasmic reticulum that contains beside the CYPs also another enzyme,
NADPH:cytochrome P450 reductase (POR), and cytochrome b5 accompanied with its
NADH:cytochrome b5 reductase. Via the activation of molecular oxygen, this multienzyme
system catalyzes the monooxygenation of a variety of xenobiotics, including BaP [6]. The
oxygen is activated in the active center of CYPs by two electrons transferred from NADPH
and/or NADH by means of POR and cytochrome b5, respectively. Whereas POR is an
essential constituent of the electron transport chain towards CYP, the role of cytochrome b5 is
still quite enigmatic. Likewise, a potential of NADH as a donor of electrons to the CYP-
mediated reaction cycle is still not exactly known. Even though the second electron in the
CYP reaction cycle might also be provided by the system of NADH:cytochrome b5 reductase,
cytochrome b5 and NADH, there is still rather enigmatic whether this system might
participate in donation of the first electron to CYP. Therefore, here we investigated the
metabolism of BaP by rat hepatic microsomes in the presence of either NADPH or NADH.
2. MATERIAL AND METHODS
Liver microsomes of rats, in which several CYP enzymes was induced with Sudan I, BaP,
phenobarbital (PB) and pregnenoloncarbonitril (PCN), were used as the enzyme systems metabolizing BaP.
HPLC was used to separate and identify BaP metabolites [7] and the 32
P-postlabling method
to detect and quantify BaP-derived DNA adducts [6].
3. RESULTS AND DISCUSSION
Rat hepatic microsomes, isolated from both uninduced animals and rats, in which expression
of individual CYPs were increased by their inducers, oxidized BaP to eight metabolites
separated by HPLC. They were identified to be BaP-9,10-dihydrodiol, a metabolite Mx, the
structure of which has not been identified as yet, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol,
BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol. These results correspond to those
found in earlier studies reporting that these metabolites were formed by CYP1A1 in a
combination with microsomal epoxide hydrolase (mEH) [2]. Of the CYP enzymes,
CYP1A1/2 and 1B1 were induced by their inducers Sudan I and BaP, CYP2B1/2 and 2C were
induced by PB and the CYPs of a 3A subfamily by PCN. Interestingly, the used rat hepatic
microsomes formed in the presence of NADH the same BaP metabolites as microsomes in the
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presence of NADPH. However, the levels of individual BaP metabolites differ depending on
CYP inducers used.
The amounts of metabolites generated by hepatic microsomes of uninduced rats and those of
induced by PCN were significantly lower when NADH was used as a cofactor of the
microsomal cytochrome P450-dependent system instead of NADPH. However, when
CYP1A1/2 and 1B1 were induced by their inducers Sudan I and BaP or CYP2B1/2 and 2C by
PB, the amounts of BaP metabolites were comparable both in the presence of NADPH and
NADH. Of BaP metabolites, the microsomes of rats exposed to Sudan I and/or BaP inducing
CYP1A1/2 and 1B1 formed the highest amounts of the activation metabolites BaP-7,8-
dihydrodiol generating dG-N2-BPDE adduct and BaP-9-ol that is a precursor of 9-hydroxy-
BaP-4,5-epoxide, which form another adduct in DNA. These results suggest that these
enzymes are most efficient in metabolic activation of BaP leading to formation of BaP-DNA
adducts. Indeed, rat CYP1A1 expressed in SupersomesTM
generated two BaP-DNA adducts,
both in the presence of NADPH and NADH and the levels of these adducts were increased by
addition of cytochrome b5 to the system. These microsomes also most efficiently oxidize BaP
to a detoxification metabolite, BaP-3-ol, in the presence of either cofactor. Rat liver
microsomes, in which CYP2B1/2 and 2C were induced by PB, were the most active
enzymatic system forming either in the presence of NADPH or NADH BaP-4,5-dihydrodiol,
the BaP metabolite that is generated by other tested microsomes as a minor product.
4. CONCLUSION
The results found in this work demonstrate that the microsomal enzymatic systems of rat liver
are capable of oxidizing BaP to its metabolites that both detoxify this carcinogen and form
BaP-DNA adducts in the presence of NADPH or NADH in vitro. They also indicate that
NADH in these microsomal systems can act as a sole electron donor both for the first and
second reduction of CYPs in their reaction cycle.
5. ACKNOWLEDGEMENT
The work has been supported by grants 15-02328S and UNCE 204025/2012.
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6. REFERENCES
[1] IARC: IARC Monographs of Evaluation of Carcinogens. Risk of Chemicals for Human, 92 (2010), 1-853
[2] Baird WM, Hooven LA, Mahadevan B: Environmental and Molecular Mutagenesis, 45 (2005), 106–114
[3] Hamouchene H, Arlt VM, Giddings I, et al.: BMC Genomics, 12 (2011), 333
[4] Phillips DH, Venitt S: International Journal of Cancer, 131 (2012), 2733-2753
[5] Fang A.H., Smith W.A., Vouros P., et al.: Biochemical and Biophysical Research Communication, 281
(2001), 383-389
[6] Stiborová M., Moserová M., Cerná V., et al.: Toxicology, 318 (2014), 1-12
[7] Indra R., Moserova M., Sulc M., et al.: Neuro Endocrinology Letters, 34 Suppl. 2 (2013), 55-63
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FABRICATION OF NANOPOROUS ALUMINA MEMBRANES FOR
ELECTROCHEMICAL SENSORS
Hana KYNCLOVÁ1,3*
, Jiří SMILEK2, Petr SEDLÁČEK
2, Jan PRÁŠEK
1,3, Martina
KLUČÁKOVÁ2, Jaromír HUBÁLEK
1,3
1 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of
Technology, Technická 3058/10, 616 00 Brno, Czech Republic
2 Faculty of Chemistry, Brno University of technology, Materials Research Centre CZ.1.05/2.1.00/01.0012,
Purkyňova 118, 612 00 Brno, Czech Republic
3 Central European Institute of Technology, Technická 3058/10, 616 00 Brno, Czech Republic
Abstract
Nanoporous alumina membrane is useful material for development of electrochemical sensors
including filtration and molecule sorting. Due to this fact, membranes with different
morphology were prepared and their morphology was studied.
1. INTRODUCTION
Membranes made of nanoporous anodic alumina oxide (AAO) represent a promising tool for
(bio) molecules sorting, filtration and purification of substances before their subsequent
detection by suitable methods. AAO is unique self-assembly porous material with relatively
low cost production. AAO is electrically non-conductive, hard, hydrophilic, chemically stable,
bioinert and biocompatible [1-3]. Alumina membranes obtained using anodization method,
have hexagonally arranged nanopores with diameter in range from 4 nm to 250 nm and
thickness from 1 µm to tens of micrometers. Nanopores are uniform, straight and
perpendicular to the surface. Dimensions of nanopores are easily controllable by changing of
experimental condition (eg. applied voltage, current density, time of anodization, type of
electrolyte) [4].
Due to above mentioned facts, nanoporous alumina membranes are used in various
applications including separation of substances which are undesirable to reach the electrode
from the detection system therefore placed behind the membrane [5]. Furthermore, the
membranes are frequently modified with different biomolecules to sort the molecules
specifically [6, 7]. In the next studies, alumina membranes are covered by conductive
materials which serve as detection electrodes [8, 9]. Transport properties (size and charge of
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diffusing molecules, rate of diffusion) of alumina membranes were studied generally using
various ions [10, 11], crystal violet dye [12] or tritiated water [13]. In our work, the various
types of alumina template were prepared. The prepared membranes will be subsequently
studied in terms of their permeability for various substances.
2. MATERIAL AND METHODS
Fabrication of nanoporous alumina
For purpose of fabrication alumina membranes were used: aluminium foil (99.999%,
Goodfellow, UK), sulphuric acid (H2SO4, 96% p.a., Penta), oxalic acid ((COOH)2, p.a. Penta,
CZ), ethanol (p. a., Penta, CZ), perchloric acid (HClO4, 70% p.a., Penta CZ) , phosphoric acid
(H3PO4, 84% p.a., Penta CZ), chromium trioxide (CrO3, p., Penta, CZ), chloric acid (HCl,
35% p.a., Penta CZ), copper chloride dihydrate (CuCl2.2H2O, p.a. Penta CZ). Deionized water
(18.2 M ) was obtained from Millipore RG system MilliQ (Millipore Corp., USA). All
chemicals were used as purchased without any purification.
The first part of alumina membrane production is so called pretreatment when a high purity
aluminium foil is polished in mixture of ethanol and perchloric acid under the potential of
20 V and temperature of 4°C for 90 seconds. The next part of fabrication is the first step of
anodization. The membranes were prepared in two types of acidic solution under the suitable
potential: 1 M sulphuric acid under the potential of 20 V and 0.3 M oxalic acid under the
potential of 40 V and 60 V respectively. The anodized part of surface is etched away in
mixture of 4.2% phosphoric acid and 3% w.t. of chromium oxide and then the second step of
anodization is performed under the same experimental conditions. The time of the second
anodization controls the thickness of alumina membrane. The last part of fabrication process
is so called postreatment. This process includes removing of non-anodized aluminium in 17%
chloric acid with addition of copper chloride dihydrate. The final step of membrane
production is etching of barrier layer created on the interface between alumina layer and
aluminium. The barrier layer is dissolved in mixture of phosphoric acid and chromium oxide.
The etching is controlled optically and takes approximately 4 minutes. All obtained
membranes were optically checked by scanning electron microscope (Tescan MIRA II,
Tescan, CZ).
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3. RESULTS AND DISCUSSION
Nanoporous alumina membranes were made by two step electrochemical anodic oxidation
under the different experimental conditions described above. In this way, membranes with
various pore diameter and thickness have been obtained. All fabricated alumina membranes
were characterized by scanning electron microscope to determine morphology of membrane
surface. The anodization in 1 M sulphuric acid under the 20 V provides nanopores with
diameter of about 30 nm and anodization in 0.3 M oxalic acid provides nanopores with
diameter of about 70 nm (Figure 3). The thickness of nanoporous alumina membranes is
dependent on time of the second anodization. The fabricated membrane will be characterized
in the term of their permeability.
Figure 3.: Nanoporous alumina membrane anodized in 0.3 M (COOH)2 (left) and 1 M H2SO4 (right)
4. CONCLUSION
Nanoporous alumina membranes with different aspect ratio were fabricated. Subsequently,
experimental setup will be designed and diffusion properties of membranes will be studied
and compared.
5. ACKNOWLEDGEMENT
The work has been supported by project no. FEKT-S-14-2300 A new types of electronic
circuits and sensors for specific applications and project no. FCH/FEKT-J-15-2663
Development of sensors based on nanoporous membranes with controlled ion permeability.
500 nm 500 nm
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6. REFERENCES
[1] Md Jani A M, Losic D and Voelcker N H.: Progress in Materials Science, 58 (2013), 5, 636-704
[2] Santos A, Kumeria T and Losic D.: TrAC Trends in Analytical Chemistry, 44 (2013), 0, 25-38
[3] (2012), Nanofabrication: Techniques and Principles, Springer,
[4] Poinern G E J, Ali N and Fawcett D.: Materials, 4 (2011), 3, 487-526
[5] Romero M R, Ahumada F, Garay F, et al.: Analytical Chemistry, 82 (2010), 13, 5568-5572
[6] de la Escosura-Muniz A, Chunglok W, Surareungchai W, et al.: Biosensors & Bioelectronics, 40
(2013), 1, 24-31
[7] Singh M and Das G: Journal of applied chemistry, 7 (2014), 1, 17-34
[8] Deng J J and Toh C S: Sensors, 13 (2013), 6, 7774-7785
[9] Cheow P-S, Ting E Z C, Tan M Q, et al.: Electrochimica Acta, 53 (2008), 14, 4669-4673
[10] Bluhm E A, Bauer E, Chamberlin R M, et al.: Langmuir, 15 (1999), 25, 8668-8672
[11] Bluhm E A, Schroeder N C, Bauer E, et al.: Langmuir, 16 (2000), 17, 7056-7060
[12] Kipke S and Schmid G: Advanced Functional Materials, 14 (2004), 12, 1184-1188
[13] Romero V, M.I.Vázques and Canete S: The journal of physical chemistry, 117 (2013), 25513-25518
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ON THE ANODIZING BEHAVIOUR OF ALUMINIUM IN CITRIC ACID
ELECTROLYTES
Tomáš LEDNICKÝ*, Alexander MOZALEV
Central European Institute of Technology (CEITEC), Brno University of Technology, Technická 3058/10,
616 00 Brno, Czech Republic
Abstract
Anodizing behaviour and porous anodic oxide growth on aluminium in citric acid electrolytes
is of considerable interest given by the high formation voltage (exceeding 300 V). In this
work we have studied the voltage-time responses during porous anodizing of aluminium foils
in 0.05 M citric acid and the impact on the anodizing behavior of various pre-treatments
involving thermal annealing, electrochemical polishing and their combination. The findings
revealed that the surface morphology and crystal structure of aluminium foils, both being
affected by the pre-treatments, greatly impact the anodizing behaviour of aluminium,
reflecting the features of pore nucleation and growth at the commencement and steady-state
period of anodizing.
1. INTRODUCTION
Anodic oxidation of aluminium and the growth of porous anodic alumina (PAA) films have
been intensively investigated over the last few decades resulting in a number of commercial
and potential applications of PAA mostly as templates for hosting various nanomaterials, such
as metals, dielectrics and semiconductors. PAA having self-ordered, versatile, honeycomb-
like cellular structure have also been used as a nanostructured support for increasing the
surface area or the surface-to-volume ratio of active layers employed in chemical sensors,
electrical capacitors, rechargeable batteries, solar cells, etc. [1, 2]. PAA formed in citric acid
electrolytes are of particular interest owing to the high forming voltages, exceeding 300 V,
leading to the formation of oxide cells approaching the micrometer size. However, a
reproducible anodic process allowing for a steady-state growth of PAA films in citric acid has
been a challenge due to the difficulty to establish the right balance between the technological,
electrical and electrolytic conditions for film nucleation and growth.
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In this work we have studied the effect of pre-anodizing treatments such as high temperature
annealing and electrochemical polishing on pore nucleation and growth through monitoring
the voltage-time behaviour during galvanostatic anodization of aluminium in a citric acid
aqueous electrolyte.
2. MATERIAL AND METHODS
An aluminium foil of 99.999 % purity and 100 µm thickness (Goodfellow) was used as the
initial material. Four types of treatments were applied to samples, cut from the foil to square
pieces of about 2 cm × 2 cm: annealing, electrochemical polishing and a combination of
annealing and electropolishing. All samples were washed in ethanol, acetone and distilled
water. Then, some of them were annealed in a vacuum at 550 °C for 5 h to allow the material
to relax and recrystallize. The electrochemical polishing was done in a mixture of perchloric
acid and ethanol (1:4 v:v) at 2-5 °C at 20 V for 1 min. After the pre-treatments, the samples
were anodized in 0.05 M citric acid (Sigma-Aldrich) aqueous solution at a constant current
density of 10 mA/cm2 at electrolyte temperature of 22.5 °C ± 0.5 °C. The voltage-time
responses were recorded until a steady-state pore growth occurred, which was associated with
reaching a relatively constant voltage behaviour of the anodizing curve [3]. The as pre-treated
and anodized samples were observed in a scanning electron microscope (SEM).
3. RESULTS AND DISCUSSION
Figure 1 shows the experimental voltage-time responses during anodizing the differently
treated aluminium foils. Three stages can be distinguished on each curve reflecting
Figure 4. Experimental voltage-time responses during galvanostatic anodizing of differently treated aluminium
foils in 0.05M citric acid at 10 mA/cm2.
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differences in pore nucleation and growth [3]. It is seen that the response of the
electrochemically polished sample (designated as “raw polished”) is shifted to higher voltages
compared with the as rolled sample (designated as “raw”). The shift becomes more evident at
stage I, which results from the phenomenon of hindering the process of pore nucleation due to
the treatment. This behaviour is associated with fewer defect sites on the polished sample
surface, which may work as the preferable centres for field-assisted dissolution and pore
nucleation and growth. Furthermore, at the steady-state pore growth on the raw polished
sample (stage III), the voltage is higher than that for the raw sample, which reflects the
growth of pores having relatively larger cell sizes, a thicker barrier layer and a smaller pore
population density [3]. This behaviour is related to pore nucleation when a smaller area is
anodized; thus pores grow locally under a larger effective current density.
The anodizing behaviour of the annealed foil is very similar to that of the raw foil during
stage I and differs mostly at the steady-state period. The first part demonstrates that a pore
nucleation strongly depends on the surface roughness, which is the case for both samples.
Small deviations may result from the increased chemical stability of the annealed sample but
still the defect sites are comparable for both samples. At stage II, the voltage does not
decrease like in the previous cases, and this implies that the pore population density remains
nearly the same. The reason for that may be an enhanced chemical stability of the annealed
surface leading to the suppressed oxide growth. This conclusion is further supported by the
SEM observations, showing that certain aluminium grains give a much thicker porous oxide
layer. This effect is more noticeable in the case of the “annealed polished” samples.
It should be finally noticed that anodizing of the “annealed polished” sample resulted in early
break-down and the rapid development of so-called plasma electrolytic oxidation of
aluminium (over 460 V). During stage I, mostly compact oxide layer is formed with
occasional pore initializations. This process shrinks enormously the area available for pore
nucleation and growth, until the local current density becomes high enough for the field-
assisted aluminium dissolution, and a steady-state pore growth eventually occurs (the end of
stage III).
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4. CONCLUSION
The new knowledge gained in this work is useful for growing PAA films with unique porous-
cellular nanostructures for use as templates and supporting substrates for nanostructuring
metals and dielectrics for potential capacitor and sensor applications.
5. ACKNOWLEDGEMENT
Research leading to these results was supported in part by GA ČR grant no. 14-29531S and by
CEITEC project STI-J-15-2886.
6. REFERENCES
[1] Mozalev A., Calavia R., et al.: Int. J. Hydrogen Energy, 38 (2013) 8011-8021.
[2] Kathko V., Mozalev A., et al.: J. Electrochem. Soc., 155 (2008), 7, K116-K123
[3] Surganov V. F., Gorokh G. G.: Mater. Lett., 17 (1993), 3-4, 121-124
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A CENTRAL ROLE FOR PHYTOCHELATIN IN PLANT AND
ANIMALS: A REVIEW
Olga KRYSTOFOVA1, Miguel Ángel MERLOS RODRIGO
1, Ondrej ZITKA
1,Vojtech
ADAM1,2
and Rene KIZEK1,2*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
Phytochelatins (PCs) are thiols formed in post-translational synthesis. They were firstly
described in yeasts Schizosaccharomyces pombe. Subsequently their presence was monitored
in plants, microorganisms, but also in many animal species. It is well known, that in plants
PCs exhibit significant function in manner of chelating of metals. Although presence of genes
encoding PCs was confirmed in a few animal species, their function in these organisms was
not satisfactorily elucidated. Some studies revealed that PCs in animal species are closely
linked with detoxification processes in similar way as in plants. It was also shown that thiols
in invertebrates can utilized as the biomarkers of heavy metals contamination
1. PHYTOCHELATINS AND PHYTOCHELATIN SYNTHASE
Higher plants, algae, certain yeasts and animals respond to heavy metals by synthesizing
phytochelatins (PCs) and related cysteine-rich polypeptides. Phytochelatin synthases are γ-
glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy
metal-binding PCs [1, 2]. PCs, cysteine-rich peptides, are produced from glutamine, cysteine
and glycine. The general structure of PCs is (c-Glu-Cys)n-Gly, with increasing repetitions of
the dipeptide Glu-Cys linked through a c-carboxylamide bond (Fig. 1), where n can range
from 2 to 11, but is typically no more than 5 [3]. Except glycine, also other amino acid
residues can be found on C-terminal end of (γ-Glu-Cys)n peptides. Ser, Glu, Gln and Ala are
often found on its place in some plant species, and they are assumed to be functionally
analogous and synthesised via essentially similar biochemical pathway. The use of an
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analytical technique able to detect compounds specifically, for example mass spectrometry, is
therefore required.
Figure 1.: The general structure of PCs and steps for synthesized from GSH through a PC synthase in response
to high concentrations of toxic metals.
2. PHYTOCHELATINS IN MICROORGANISMS
Interestingly, although PC(n=2) has been described in the yeast S. cerevisiae, there is no
homologue of the PC-synthase genes in the S. cerevisiae genome. An alternative pathway for
PCs biosynthesis in S. pombe has been proposed, however, and it may be that this pathway
functions in S. cerevisiae [4]. One study showed that the two vacuolar serine
carboxypeptidases are responsible for PC synthesis in S. cerevisiae. The finding of a PCS-like
activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.
3. PHYTOCHELATINS IN PLANTS
Chelation and sequestration of metals by particular ligands are also mechanisms used by
plants to deal with metal stress. Naturally hyperaccumulating plants do not overproduce PCs
as part of their mechanism against toxic metals. Several studies of plants that overexpressed γ-
glutamyl-cysteine synthetase or transgenic plants expressing bacterial γ-glutamyl-cysteine
synthetase evaluated its effect on metal tolerance based on the assumption that higher levels
of GSH and PCs will lead to more efficient metal sequestration. Arabidopsis thaliana showed
that Cd is immediately scavenged by thiols in root cells, in particular PCs, at the expense of
GSH. At the same time, a redox signal is suggested to be generated by a decreased GSH pool
in combination with an altered GSH:GSSG ratio in order to increase the antioxidant capacity
[5]. Overexpression of PCs synthetize in Arabidopsis led to 20-100 times more biomass on
250 and 300 μM arsenate than in the wild type. Gamma-glutamyl cysteine, which is a
substrate for PC synthesis, increased rapidly, after arsenate or cadmium exposure.
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4. PHYTOCHELATINS IN ANIMALS
PCs proteins have been broadly described and characterized in plants, yeasts, algae, fungi and
bacteria, as well as nematodes and trematodes [6]. PC synthase genes are also present in
animal species from several different phyla. PCs synthesis appears not to be transcriptionally
regulated an animal [7]. Originally thought to be found only in plants and yeast, PC synthase
genes have since been found in species that span almost the whole animal tree of life.
Biochemical studies have also shown that these PCS genes are functional: the Caenorhabditis
elegans PC synthase produces PCs when it is expressed in an appropriate host, and knocking
out the gene increases the sensitivity of C. elegans to cadmium [8]. Several studies have since
measured PCs by direct biochemical analysis of C. elegans tissue extracts, and found that
cadmium exposure did indeed increase PCs levels in C. elegans. PC2, PC3, and PC4 have all
been found, with PC2 the highest concentration. Therefore, these studies showed conclude
that PCs production plays a major role in protecting C. elegans against cadmium toxicity. PC2
and PC3 were increased in autochthonous Lumbricus rubellus populations sampled from
contaminated sites. It is important to say that MTs are widely established as a key metal
detoxification system in animals, even though they certainly have many other biological
functions as well. As yet, there is very little known about how MTs and PCs may complement
each other for dealing with toxic metals.
5. ACKNOWLEDGEMENT
The work has been supported by project MENDELUCZ.1.07/2.3.00/30.0017
6. REFERENCES
[1] Vatamaniuk O K, Mari S, Lu Y P, et al., J. Biol. Chem., 275 (2000), 31451-31459.
[2] Rea P A, Physiol. Plant., 145 (2012), 154-164.
[3] Pivato M, Fabrega-Prats M, Masi A, Arch. Biochem. Biophys., 560 (2014), 83-99.
[4] Hayashi Y, Nakagawa C W, Mutoh N, et al., Biochemistry and Cell Biology-Biochimie Et Biologie
Cellulaire, 69 (1991), 115-121.
[5] Jozefczak M, Keunen E, Schat H, et al., Plant Physiol. Biochem., 83 (2014), 1-9.
[6] Rigouin C, Vermeire J J, Nylin E, et al., Mol. Biochem. Parasitol., 191 (2013), 1-6.
[7] Liebeke M, Garcia-Perez I, Anderson C J, et al., Plos One, 8 (2013).
[8] Bundy J G, Kille P, Liebeke M, et al., Environ. Sci. Technol., 48 (2014), 885-886.
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STUDY OF CHARACTERIZATION MAMMALIAN
METALLOTHIONEINS BY MALDI-TOF/TOF AND
ELECTROCHEMICAL METHOD
Miguel Ángel MERLOS RODRIGO1, Jorge MOLINA LOPEZ
2, Ondrej ZITKA
1, Rene
KIZEK1*
1 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
2 Department of Physiology, Institute of Nutrition and Food Technology, University of Granada, Avd. Del
Conocimiento S/N Biomedical Research Centre, Health Campus, Granada, Spain, European Union
Abstract
Zinc is one of the most abundant and important metal ions in biology. Mammalian
metallothionein (MTs) have two domain: alpha and beta. They play a crucial role in storing
and donating Zn2+
ions to target metalloproteins and have been implicated in several diseases.
Here, we show a novel and fast method for characterization the MTs and MTs-Zn complexes
from different mammalian MTs isoforms by electrochemical method and MALDI-TOF/TOF
mass spectrometry.
1. INTRODUCTION
Zinc is a drug in the prevention and management of many diseases: inflammation, necrosis
and cancer [1, 2]. Mammalian MTs comprise a Zn(3)Cys(9) cluster in the beta domain and a
Zn(4)Cys(11) cluster in the alpha domain. They play a crucial role in storing and donating
Zn2+
ions to target metalloproteins and have been implicated in several diseases [3]. MT
induction and zinc administration are novel strategies to sensitize colorectal cancer cells to
presently utilized chemotherapeutic agent [4]. Here, we show method for characterization the
MTs and MTs-Zn complexes from different mammalian MTs isoforms by electrochemical
method and MALDI-TOF/TOF mass spectrometry.
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2. MATERIAL AND METHODS
Isolation MTs and plasmids constructions
The human MT3 and MT2A genes were cloned by pRSET-B vector, for expression MTs in
BL21 (DE3) Chemically Competent E. coli strain. The rabbit MT2 protein was isolated from
rabbit liver. The isolation was homogenised on ice with 10 mM Tris–HCl buffer and
vortexed. In the end purification of protein was done by fast protein liquid chromatography.
The Matrix-Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometric
(MALDI-TOF/TOF)
MTs were analyzed by MALDI-TOF-MS (ultraflex III instrument, Bruker Daltonik,
Germany). The matrix used was 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-
hydroxycinnamic acid (HCCA) prepared in TA30. 1µl was applied on the target and dried
under atmospheric pressure and ambient temperature. A mixture of peptide calibrations
standard (Bruker) was used to externally calibrate the instrument.
Adsorptive Transfer Stripping (AdTS) Differential Pulse Voltammetry (DPV)
MT was measured using AdTS DPV. The supporting electrolyte was sodium chloride (0.5 M
NaCl, pH 6.4). DPV parameters were as follow: the initial potential of -1.5 V, the end
potential 0 V, the modulation time 0.057 s, the interval 0.2 s, the step potential of 1.05 mV/s,
the modulation amplitude of 25 mV.
3. RESULTS AND DISCUSSION
In our study, 2.5-DHB showed high increased of signal intensity (a.u) than HCCA when
increased the concentration of rMT2 (Fig.1.A). The main observed signals for MTs shown in
Figure.1 were assigned as follows: [rMT2]c+ (m/z 6210.65), [6xHis-tag-hMT2A]
+ (m/z
7280.15) and [hMT3]+ (m/z 6909.92). We assume the signal peaks labelled ZnT’ and ZnT,
and CuT, are due to the reduction of the zinc and copper complexed with the MT, possibly in
two different forms of complexation from electrochemical reactions proceeding between MT
and heavy metals. We found out that MT signals were influenced by different concentrations
of NaCl. ZnT’, MT(Zn) and MT(Cu) signals changed according to different ionic strength
markedly, whereas MT(Zn) signal measured in the presence 0.1 M NaCl was very low in
comparison with 0.5 M NaCl. Finally we evaluate the influence of pH on the electrochemical
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detection of MT. We observed influence of changes of supporting electrolyte pH (0.5 M
NaCl) within the range from 6.5 to 7.5 (adjusted by additions of HCl and/or NaOH) on
electrochemical signal of MT. In the case of increasing concentration of Zn(II) in presence to
MT, five peaks appeared (ZnT, CdT, MT(Zn), MT(Cu) and CuT) corresponding to MT heavy
metals complexes formed. On the other hand, increasing Zn(II) concentrations caused linear
rise of MT(Zn) peaks height.
Figure 1.: Spectrum of rMT2 and graphs of signal intensity of different concentration of rMT2 in 2,5-DHB and
HCCA matrix (A). Photo of MTs crystals on target plate with 2,5-DHB (B) and HCCA matrix (C) and spectrum
in both matrix. Spectrum 6xHist-tag-hMT2A (D), and hMT3 (E). Typical DP voltammograms of 2uM MT
Rabbit Liver measured in the presence of 0.5M NaCl, pH 6.5. Dependences of heights of ZnT’, ZnT, MT(Zn),
CuT and MT(Cu) signals on accumulation times (F) of 120s, 240s, 360s, on concentration of electrolyte (G)
(0.1M, 0.3M, 0.5M) and on pH (H) (6.5, 7.0, 7.5), expresed in relative percentage peaks and peak height (nA).
4. CONCLUSION
Data included in this work highlight the potential of DPV to be used, in combination with
other analytical and MALDI-TOF/TOF, for monitoring structural differences among MTs and
metal-MTs complexes.
5. ACKNOWLEDGEMENT
The work has been supported by FA COST Action TD 1304.
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6. REFERENCES
[1] Vasto S, Candore G, Listi F, et al., Brain Res. Rev., 58 (2008), 96-105.
[2] Desouki M M, Geradts J, Milon B, et al., Molecular Cancer, 6 (2007).
[3] Babu C S, Lee Y-M, Dudev T, et al., Journal of Physical Chemistry A, 118 (2014), 9244-9252.
[4] Arriaga J M, Greco A, Mordoh J, et al., Molecular Cancer Therapeutics, 13 (2014), 1369-1381.
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CORE/SHELL QUANTUM DOTS AS FLUORESCENT LABELS OF
BIOMOLECULES
Ana MIHAJLOVIĆ1,2*
, Jana PEKÁRKOVÁ1,2
, Jaromír HUBÁLEK1,2
1 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of
Technology, Technická 3058/10, 616 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technická 3058/10, 616 00 Brno,
Czech Republic
Abstract
CdTe quantum dots (CdTe) capped with glutathione (GSH), thioglycolic (TGA) or
mercaptopropionic acid (MPA) were prepared in aqueous phase, and used for synthesis of
colloidal core/shell CdTe/ZnS QDs. Core/shell QDs were used for conjugation with bovine
serum albumin (BSA) via different cross-linkers (EDC/NHS, EDC). QDs as well as QDs-
BSA conjugates were characterized via UV-Vis spectroscopy and it was found that with
increasing concentration of BSA fluorescence intensity of QDs decreased.
1. INTRODUCTION
Up to date, many scientific studies have yielded different approaches of synthesis,
modification and conjugation of QDs. QDs synthesized through aqueous route exhibit low
toxicity, excellent biological compatibility and stability [1]. Many studies have addressed the
issue of surface defects and it has been observed that these defects can be minimized or
entirely eliminated by using capping agents such as TGA, MPA, GSH and others.
Conjugation with biomolecules can be achieved through covalent coupling using cross-
linkers, which are molecules that through their carboxyl groups enable more successful bond
making between QDs and its target. The most frequently used cross-linkers to achieve
covalent conjugation are EDC (N-(3- Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride) and NHS (N-Hydroxysuccinimide [1-3].
In this paper, different water soluble CdTe QDs capped with GSH, TGA or MPA and
core/shell CdTe/ZnS QDs were prepared and used for detection of BSA. QDs and QDs-BSA
conjugates were characterized via UV-Vis spectroscopy.
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2. MATERIAL AND METHODS
Synthesis of CdTe QDs capped with GSH, MPA or TGA
To obtain CdTe-GSH QDs, CdCl2 solution (0.04 mol/l, 4ml) was diluted to 46 ml in one-
necked flask and then GSH (300 mg), sodium citrate dihydrate (100 mg), Na2TeO3 (0.01
mol/l, 4 ml), NaBH4 (50 mg) were added under vigorous stirring. Solution was then refluxed
at 95°C for 3 h [1].
To obtain CdTe-MPA QDs, CdCl2 solution (91.6 mg) was diluted to 50 ml in one-necked
flask and sodium citrate dihydrate (200 mg) was added followed by addition of MPA (52 µl).
The pH of the solution was adjusted to 10.5 using NaOH (1 mol/l), followed by addition of
Na2TeO3 (22.15 mg) and NaBH4 (50 mg) under vigorous stirring. Solution was then refluxed
at 95°C for 4 h [2].
To obtain CdTe-TGA QDs, CdCl2 solution (183 mg) was diluted to 48 ml in one-necked flask
and TGA (104 µl) was added followed by adjustment of pH to 10.5 using NaOH (1 mol/l).
Then sodium citrate dihydrate (50 mg), Na2TeO3 (0.01 mol/l, 2 ml) and NaBH4 (20 mg) were
added under vigorous stirring. Solution was then refluxed at 95°C for 4 h [3].
Synthesis of CdTe/ZnS QDs
To obtain CdTe/ZnS QDs, CdTe (40 mg) capped with GSH, MPA or TGA were diluted to 50
ml in one-necked flask followed by addition of ZnCl2 (6.8 mg) and GSH (61.3 mg) were
added under vigorous stirring. The pH of the solution was adjusted to 8 using NaOH (1 mol/l)
and solution was then refluxed at 95°C for 3 h [4].
Preparation of QDs-BSA conjugates via EDC/NHS
Briefly, to the solution of core/shell CdTe/ZnS QDs (200 µl, 0.1 mg/ml) EDC (200 µl, 50
mmol/l) and NHS (200 µl, 5 mmol/l) were added and solution was then incubated at 32°C for
30 min. Then, BSA (200 µl; 0, 0.0005, 0.002, 0.005, 0.05 mg/ml) was added to the solution
and incubated at 32°C for 2 h while shaking [5].
Preparation of QDs-BSA conjugates via EDC
Briefly, to the solution of core/shell CdTe/ZnS QDs (250 µl, 0.1 mg/ml) BSA (250 µl; 0,
0.05, 0.5, 1 a 1.5 mg/ml) and EDC (57 µl, 10 mg/ml) were added. The solution was then
incubated at room temperature for 2 h [6].
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3. RESULTS AND DISCUSSION
UV-Vis spectroscopy results showed, that the highest intensity of fluorescence is in the case
of CdTe-MPA/ZnS QDs (95277 CPS at 626 nm) while the lowest intensity of fluorescence is
in the case of CdTe-GSH/ZnS QDs (10525 at 512 nm). Parameter full width at half maximum
(FWHM) was also analyzed, because it indicates the uniformity of size distribution of QDs.
FWHM is 86 nm, 42 nm and 86 nm in the case of CdTe-MPA/ZnS QDs, CdTe-TGA/ZnS
QDs and CdTe-GSH/ZnS QDs. This indicated that the best uniformity of size distribution of
QDs is in the case of CdTe-TGA/ZnS QDs.
From Figure 1, based on Stern-Volmer equation, it is noticeable, that in case where EDC
alone was used as cross-linker, the highest quenching effect was noticeable in the case of
CdTe-MPA/ZnS QDs while in case of CdTe-GSH/ZnS QDs the quenching effect was the
least obvious. In the case where conjugation is achieved via combination of EDC and NHS,
the highest quenching effect was evident in the case of CdTe-MPA/ZnS QDs while in case of
CdTe-GSH/ZnS QDs the quenching effect was the least apparent.
Figure 1: Stern-Volmer plots for quenching of QDs fluorescence by different concentrations of BSA.
Conjugation via EDC/NHS (a), via EDC (b).
4. CONCLUSION
In this paper, a method for synthesis of colloidal core and core/shell QDs and three methods
for preparation of core/shell QDs-BSA conjugates were described. QDs and QDs-BSA
conjugates were analyzed via UV-Vis spectroscopy. The results obtained illustrated
quenching effect of different concentrations of BSA on fluorescence intensity of QDs.
(a) (b)
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5. ACKNOWLEDGEMENT
This work has been supported by the National Sustainability Program under grant LO1401
and Grant Agency of the Czech Republic under the contract GP 13-20303P. For the research,
the infrastructure of the SIX Center was used.
6. REFERENCES
[1] Zhu Y, Chen M, Cooper M H, Lu M Q G and Xu P Z: Journal of Colloid and Interface Science, 390 (2013),
1, 3-10
[2] Long Z, Jia J, Wang S, Kou L, Hou X and Sepaniak M: . Microchemical Journal, 110 (2013), 0, 364-369
[3] Wang J and Han H: Journal of Colloid and Interface Science, 351 (2010), 1, 83-87
[4] Liu Y F and Yu S J: Journal of Coloid and Interface Science, 351 (2010), 1, 1-9
[5] Chopra A, Tuteja S, Sachdeva N, Bhasin K K, Bhalla V and Suri C R: Biosensors and Bioelectronics, 44
(2013), 0, 132-135
[6] Tian J, Liu R, Zhao Y, Xu Q and Zhao S: Journal of Colloid and Interface Science, 336 (2009), 2, 504-509
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BENZO[A]PYRENE, ELLIPTICINE AND 1-PHENYLAZO-2-
NAPHTHOL INDUCE EXPRESSION OF CYTOCHROME B5 IN RATS
Marie STIBOROVÁ1*
, Michaela MOSEROVÁ1, Iveta MRÍZOVÁ
1, Helena DRAČÍNSKÁ
1,
Věra ČERNÁ, EVA FREI2, Volker M. ARLT
3.
1 Department of Biochemistry, Faculty of Science, Charles University, Prague, Albertov
2030, 128 40 Prague 2, Czech Republic
2 Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer
Research Center (DKFZ), Im Neuenheimer Feld 280, 69 120 Heidelberg, Germany
3 Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and
Health, King’s College London, London, United Kingdom
Abstract
Expression of cytochrome b5 the protein influencing activity of several cytochrome P450
(CYP) enzymes, was found to be induced at the protein and mRNA levels by exposure of rats
to three aryl hydrocarbon receptor ligands, benzo[a]pyrene (BaP), 1-phenylazo-2-naphtol
(Sudan I) and ellipticine. Because this protein modulates activities of CYPs such as CYP1A1
and 3A4 oxidizing these compounds to genotoxically and/or pharmacologically efficient
metabolites, its expression levels determines their biological activities.
1. INTRODUCTION
Cytochromes b5 are heme proteins, which are capable of accepting and transferring a single
electron [1]. One of cytochromes b5, which is located in the membrane of endoplasmic
reticulum (microsomal cytochrome b5), is involved in fatty acid desaturation, cholesterol and
plasmalogen biosyntheses as well as in various hydroxylation reactions catalyzed by mixed
function oxidase system [2,3]. It can accept an electron from either NADH:cytochrome b5
reductase or NADPH:cytochrome P450 (CYP) reductase [3,4] and then reduced cytochrome
b5 transfers this electron to CYPs and other enzymes. The role of microsomal cytochrome b5
in catalytic function of CYPs has not been fully understood yet. Cytochrome b5 has been
shown to be able to stimulate, inhibit or have no effect on CYP mediated reactions (for a
review, see [2-4]). One of hypotheses trying to explain the influence of cytochrome b5 on
CYP reactions suggests a role of cytochrome b5 in a direct transfer of the second electron to
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the CYP enzyme, which is considered to be the rate limiting step in the catalytic cycle of the
CYP monooxygenase reaction [4]. The electron transfer from reduced cytochrome b5 to CYP
is faster than the input of electron from NADPH:CYP reductase [5]. Another possible
mechanism of the cytochrome b5 action is the formation of a complex between cytochrome b5
and CYP, which can receive two electrons from NADPH:CYP reductase in a single step, one
for reduction of CYP and another for that of cytochrome b5 [3]. While CYP without
cytochrome b5 has to undergo two separate interactions with NADPH:CYP reductase to
complete one catalytic cycle, in the case of the presence of cytochrome b5, only one single
interaction of complex of CYP and cytochrome b5 with NADPH:CYP reductase is sufficient;
cytochrome b5 provides the second electron to CYP promptly after oxygen binding.
Interaction of cytochrome b5 with CYP may also induce conformational changes in CYP
proteins leading to breakdown of oxygenated hemoprotein complex with substrates to
products. This hypothesis is based on findings showing that not only holoprotein of
cytochrome b5, but also its apo-form (devoid of heme), which is not capable of electron
transfer, can contribute to stimulation effects [3-5].
It is clear from such investigations that expression levels of cytochrome b5 in cells are crucial
for efficiencies of several CYPs to oxidize xenobiotics. This is also true for the oxidation of
the anticancer drug ellipticine, and the carcinogens benzo[a]pyrene (BaP) and 1-phenylazo-2-
naphthol (Sudan I); their oxidation by CYP1A1 and/or 3A4, dictating their biological effects,
is strongly influenced by cytochrome b5 [6-12]. Therefore, here the effect of BaP, ellipticine
and Sudan I on expression of cytochrome b5 mRNA and protein in rats in vivo was
investigated.
2. MATERIAL AND METHODS
Male Wistar rats were treated intraperitoneally with BaP, ellipticine and Sudan I as described
previously [7-10]. Microsomes were isolated from the livers and kidneys of this animal model
[9]. The method of Western blot, employing anti-rat cytochrome b5 antibodies, was utilized to
evaluate expression of this protein. Its mRNA contents in rat liver and kidney measured using
the real-time polymerase chain reaction (RT-PCR) was also carried out [7-10].
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3. RESULTS AND DISCUSSION
Using a method of Western blotting with antibodies raised against rat cytochrome b5, the
effects of exposure of rats to BaP, ellipticine and Sudan I on mRNA and protein expression
levels of these proteins were analyzed. These compounds were found to be inducers of
cytochrome b5 in liver and kidney of exposed rats. The mechanism of such induction was
investigated in this work. It is known that beside a potency of BaP, ellipticine and Sudan I to
induce cytochrome b5, they also induced enzymes that are regulated by activation of the aryl
hydrocarbon receptor (AHR), CYP1A1 and NQO1, both at mRNA and protein levels. This
corresponds to their ability to act as AHR ligands [6,7,9-12].
Up to 10-fold increase in cytochrome b5 protein expression levels was caused by treatment of
rats with ellipticine, BaP and Sudan I. The increase in protein levels was paralleled by an
increase in mRNA expression in most cases. The results found in this work suggest that
induction of cytochrome b5 by the tested xenobiotics might be mediated by a mechanism
dependent on activation of AHR. However, additional studies investigating the binding of
tested compounds to AHR and its activation to be moved in a complex with ARNT protein to
nucleus and bound to the response element for cytochrome b5 expression should be carried
out to confirm this suggestion.
4. CONCLUSION
Employing the electromigration assays combined with immunochemical determination
(Western blot), expression of cytochrome b5 protein was found to be induced by treating rats
with AHR ligands, BaP, ellipticine and Sudan I. Since cytochrome b5 is crucial for oxidation
of these xenobiotics by CYP enzymes [8,11,12], these compounds exert concerted regulatory
control on their own pharmacological and genotoxic activities.
5. ACKNOWLEDGEMENT
The work has been supported by grants 15-02328S and UNCE 204025/2012.
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6. REFERENCES
[1] Velick SF, Strittmatter P: Journal Biological Chemismy, 221 (1956), 265-275.
[2] Vergeres G, Waskell L.: Biochemie, 77 (1995), 604-620.
[3] Schenkman JB, Jansson I: Pharmacology and Therapy, 97 (2003), 139-152.
[4] Guengerich FP:, Archives of Biochemistry and Biophysics, 440 (2005,) 204-211.
[5] Schenkman JB, Jansson I: Drug. Metabolism Review, 31 (1999), 351-364.
[6] Aimová D., Svobodová L., Kotrbová V., et al.: Drug Metabolism and Disposition, 35 (2007), 1926-1934.
[7] Arlt VM, Stiborová M, Henderson CJ, et al.: Carcinogenesis, 29 (2008), 656-665.
[8] Kotrbová V., Mrázová B., Moserová M., et al.: Biochemical Pharmacology, 82 (2011), 669-680.
[9] Vranová I, Moserová M, Hodek P, et al.: International Journal of Electrochemical Science, 8 (2013), 1586-
1597.
[10] Stiborová M, Dračínská H, Martínek V, et al.: Chemical Research in Toxicology, 25 (2013), 290-299.
[11] Stiborová M, Moserova M, Černá V, et al., Toxicology, 318 (2014), 1-12.
[12] Stiborová M, Schmeiser HH, Frei E, et al. Current Drug Metabolism, 15 (2014), 829-840.
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LEAD PENCIL GRAPHITE AS ELECTRODE MATERIAL:
STRUCTURAL AND ELECTROCHEMICAL PROPERTIES
Rudolf NAVRATIL1, Jan HRBAC
1,2, Vladimir HALOUZKA
2,3 Libuse TRNKOVA
1,4*
1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
2 Department of Analytical Chemistry, Faculty of Science, Palacky University, 17. listopadu 12, CZ-771 46
Olomouc, Czech Republic.
3 Department of Physics and Materials Engineering, Faculty of Technology, Tomas Bata University in Zlin, nam.
T.G. Masaryka 275, 76001 Zlin, Czech Republic
4 Central European Institute of Technology, Technicka 3058/10, 616 00 Brno, Czech Republic
Abstract
In recent decades, there has been a significant progress in the development of solid electrodes
leading to their use as sensors to detect various substances in electrochemical assays. The
field of carbon electrodes that attract attention because of the unique structural, mechanical
and electrochemical properties is a particular example of this trend [1].
The aim of this work is the structural and electrochemical characterization of several pencil
graphite electrodes (PeGE) from different manufacturers in relation to their electrochemical
properties. The surface quality affects mainly the adsorption of the species involved in redox
reactions and rate of electron transfer. Therefore, PeGEs were compared by using
voltammetric methods such as cyclic voltammetry (CV) with other types of graphite
electrodes which have different structures [2].
In addition to electrochemical experiments, the composition and structure of the pencil leads
were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy,
Raman spectroscopy and X-ray photoelectron spectroscopy [3].
EXPERIMENTAL
Apparatus: The measurements were performed on the AUTOLAB analyzer in connection
with a VA-Stand 663. The voltammetric cell included a three-electrode (working electrode
PeGE (Pencil Graphite Electrode – 0.5 HB Tombow, Japan, 0.5 HB Sakota, Japan, 0.5 HB
Pilot, France), a reference electrode (Ag/AgCl/3M KCl), and an auxiliary electrode (platinum
wire). SEM analysis was performed on the scanning electron microscope Mira II LMU.
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Voltammetric analysis: To activate the PeGE, we used CV method (cyclic voltammetry) with
the following setup: initial potential: 0 V, final potential: 1.4 V, step potential: 5 mV, standby
potential: 0 V. conditioning potential: 1.4 V, duration: 30 s. Activated electrodes were used
for LSV (linear sweep voltammetry) experiments. For EVLS evaluation, LSV curves were
measured at three different scan rates (200, 400 and 800 mV/s) [4]. The LSV setting was as
follows: start potential = conditioning potential: -0.15 V, duration time: 120 s, final potential:
1.4 V, step potential: 5 mV, standby potential: 0 V. For the processing of the data and as a
control device the GPES 4.9 program was applied. Electrochemical measurements were
performed in 0.1 M acetate buffer.
RESULTS AND DISCUSSION
Figure 1.: Scanning electron microscope (SEM) pictures of Tombow, Pilot, KOH-I-NOOR and Sakota leads for
four values of magnification (500, 1000, 5000 and 40000 times)
Figure 2.: Linear sweep voltammograms of Xanthine (20 µM) in the absence (A) and in the presence (B) of
Cu(II) (20 µM) in acetate buffer (pH 5.1) for recorded at different types of electrodes, scan rate = 400 mV/s.
Tombow Pilot KOH-I-NOOR Sakota
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CONCLUSION
In this work, several types of pencil graphite leads have been tested as a tool for the detection
of purines by adsorption stripping techniques in combination with LSV and EVLS. To
improve the sensitivity of detection, additions of copper (II) ions were used, which form
slightly soluble complexes Cu (I)-purine on the electrode surface by a reductive process. Also,
the comparison of the composition and structure (SEM, X-Ray, Raman) was performed and
the electrochemical properties of these electrodes were tested using voltametric experiments
with redox standards. It was found that even slight changes in the composition of the polymer
constituting the fibers leads cause noticeable differences in the detection of different types of
graphite leads. The experiments show that from the used electrodes the most appropriate are
Tombow leads. These leads have proven themselves as most suitable for detection purposes.
Compared to other studied electrodes, Tombow leads exhibited high repeatability and
reproducibility of results, but also provided the most sensitive determination of the monitored
substances. The use of pencil graphite electrodes (PeGE) appears to be a good choice for the
detection of oxidative signals of biologically important compounds, potentially leading to
low-cost, sensitive, simple and non-toxic electrochemical sensor for the qualitative and
quantitative determination of these substances in medicine or pharmacy.
ACKNOWLEDGEMENT
The work has been supported by projects: (1) Postdoc project "Employment of Newly
Graduated Doctors of Science for Scientific Excellence" (CZ.1.07/2.3.00/30.0009) (2)
KONTAKT II LH 13053 and (3) MUNI/A/0972/2013 of Ministry of Education, Youth and
Sports of the Czech Republic.
REFERENCES
[1] Kinoshita K, Carbon: electrochemical and physicochemical properties, John Wiley & Sons, New York, 1988.
[2] McCreery R. L.: Chem. Rev., 108 (2008), 2646-2687.
[3] Kariuki J. K.: J. Electrochem. Soc., 159 (2012), H747-H751.
[4] Navratil R., Jelen F., Kayran Y. U., Trnkova L.: Electroanalysis, 2 (2014), 952-961.
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CHEAP AND QUICK PRODUCTION OF MICRO AMALGAM
ELECTRODES FOR DETERMINATION OF SOIL CONTAMINATED
WITH HEAVY METAL IONS (CD(II) AND PB(II))
Lukas NEJDL1,4
, Magdalena HABOVA2, Jiri KUDR
1,4, Branislav RUTTKAY-NEDECKY
1,4,
Jindrich KYNICKY3, Lubica POSPISILOVA
2, Vojtech ADAM
1,4 and Rene KIZEK
1,4*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of Agronomy, Mendel
University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic
3 Department of Geology and Pedology, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech
Republic
4 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
Heavy metal pollution has become one of the most serious environmental problems. In this
paper, we present low-cost and rapid production of amalgam electrodes, which were used for
determination of Cd(II) and Pb(II).
1. INTRODUCTION
Voltammetry is the most commonly used electrochemical method, where the potential is
inserted on working electrode [1]. Inserted potential can be changed and thus the current
response is monitored. This basic electrochemical method is usually used for study and to
determination of substances dissolved in aqueous solutions or in organic solvents. For these
applications most commonly used electrodes are mercury, amalgams, gold, platinum and
carbon working electrodes. The amalgam electrode is alternative to mercury hanging drop
electrode (HMDE) mainly therefore amalgam electrode shows similar sensitivity as HMDE
and many other advantages. For example amalgam electrodes have a lower toxicity, easy
manipulation, can be used multiple times, miniaturizations and flow is possible (automation)
[2]. The big advantage is the ability to analyse turbid samples [3]. The production of this
electrode is cheap and fast as we show in this paper.
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2. MATERIAL AND METHODS
Chemicals and material
All metal standards including lead(II) nitrate and cadmium(II) sulphate were purchased from
Sigma-Aldrich (St. Louis, MO, USA). As electrolyte acetate buffer (0.2 M, pH 5) was
utilized.
Modification of electrolytic copper wire as working electrode (Hg/Cu-WE)
Copper wires (Thermo scientific, Cambridge, UK) were used as working electrodes after
modification. The copper wire were inserted into 0.01 M Hg(NO3)2 solution, prepared by the
dissolution of 0.086 g mercury(II) nitrate in 25 mL of acidified (5% HNO3, v/v) Milli-Q
water.
Optimization of Cd(II) and Pb(II) detection
Electrochemical detection was performed using a three electrode system. Solid Hg/Cu-WE
electrode with dimensions of 0.3 (diameter) × 10 mm (length) was used. An Ag/AgCl/3 M
KCl electrode was the reference (RE) and platinum electrode was auxiliary (CE). The
parameters of the DPV measurement were as follows: initial potential -1.1 V; end potential
-0.2 V; step potential 0.005 V; modulation amplitude 0.025 V; modulation time 0.057 s,
deposition time 90 sec and interval time 0.2 s.
3. RESULTS AND DISCUSSION
Detection was performed using a classical three-electrode system (working, reference and
auxiliary electrode). Cu wire covered with amalgam was used as working electrode (Hg/Cu-
WE) for all electrochemical measurements. Electrochemical response of 800 ng.ml-1
Cd(II)
and Pb(II) was not recorded with Cu wire, application of Cu wire coated with amalgam led to
the appearing of electrochemical signal (Fig. 1A). For all optimization experiments 800 ng.ml-
1 Cd(II) and Pb(II) were used. An interesting finding was that the unmodified Cu wire exhibits
no electrochemical response, but after immersion (1 s) into a solution of 0.01 M Hg(NO3)2
high electrochemical signals for Cd(II) and Pb(II) were recorded (Fig. 1B). The best
electrochemical response was achieved in the amalgamation time 60 s. Next optimization was
focused on monitoring of the electrochemical response of the individual elements depending
on the increasing deposition time within the range from 0 to 160 s. It has been shown that an
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increase in the deposition time caused increases in the electrochemical signals of Cd(II) and
Pb(II) (Fig. 1C). Furthermore, the influence of the HCl on the change of the electrochemical
signal was observed. It was found that 0.25 and 0.5% HCl caused a significant increase in
Cd(II) signal, Fig. 1D (red column). The electrochemical signal of Pb(II) was changed within
a 5% error, Fig. 1D (blue column). This experiment proved that Cd(II) and Pb(II) can be
detected in the soil leachate containing 1% HCL, without significant changes of the
electrochemical signal.
Figure 1.: (A) Voltammogram of 800 ng.ml-1
Cd(II) and Pb(II). (B) Dependence of the time of amalgamation
(Cu wire immersion time in the solution of 0.01M Hg (NO3)2) in the range of 0-480 s on the electrochemical
response of 800 ng.ml-1
of cadmium or lead. (C) The dependence of the deposition time (0-160s) on the
electrochemical response of 800 ng.ml-1
of both cadmium, or lead. (D) The effect of HCl concentration to change
the electrochemical signal of 800 ng.ml-1
of both cadmium, or lead.
4. CONCLUSION
In this work fast and cheap manufacture of amalgam electrodes was presented. On this type of
WE electrodes electrochemical method (differential pulse voltammetry) for the detection of
Cd(II) and Pb(II) has been optimized. This method can be used for analysis of Cd(II) and
Pb(II) in soil leachates containing 1% HCL.
5. ACKNOWLEDGEMENT
Financial support from UGP ID: 1912015 and IGA SP 2150821 are highly acknowledged.
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6. REFERENCES
[1] Davies T J, Banks C E, Compton R G, J. Solid State Electrochem., 9 (2005), 797-808.
[2] Mikkelsen O, Schroder K H, Electroanalysis, 15 (2003), 679-687.
[3] Chai C Y, Liu G Y, Li F, et al., Anal. Chim. Acta, 675 (2010), 185-190.
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SYNTHESIS OF CONJUGATED AROMATIC SYSTEMS AND THEIR
PROPERTIES
Michaela BRHELOVÁ, Hugo SEMRÁD, Markéta MUNZAROVÁ, Iveta PILAŘOVÁ,
Libuše TRNKOVÁ, Milan POTÁČEK*
Department of Chemistry, Faculty of Science, Masaryk University,Kotlářská 2, 611 37 Brno, Czech Republic
Abstract
The presented paper deals with the synthesis of conjugated systems consisted from phenyl,
thiophene or mixed phenyl and thiophene units, substituted on the ends to create molecules
with push-pull delocalized electronic orbital system. The synthesis is based upon
application of modern Suzuki-Miyaura coupling reaction. This reaction represents break down
in reactivity of halogen at aromatic hydrocarbon. Using aromatic boronic acids in the reaction
with halogen containing aromatic skeleton, in the presence of homogenous palladium catalyst
and presence of a base, biaryl compound is synthesized.
The prepared new compounds underwent examination on their physical-chemical properties.
Their UV and fluorescence spectra are presented, cyclic voltammetry was carried out, and
basic quantum chemistry calculations are submitted. Based on this data possible application is
displayed.
1. INTRODUCTION
Organic molecules consisting of various conjugated systems have found its application as
light-emitting, light absorbing and semiconducting materials [1]. Because systems with push-
pull substitution strongly effect the levels of the frontier orbitals, they are known to exhibit
narrowed energy gaps and strong dipoles due to intramolecular charge transfer. Such
materials have been of interest as long-wavelength absorbing dyes [2] and in nonlinear optics
[3].
Synthesis of such systems with substitution creating push-pull systems was carried out with
application of modern cross-coupling reactions.
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2. SYNTHESIS AND METHODS OF INVESTIGATION
Synthesis
They were prepared three lines of conjugated compounds differing in aromatic components.
The first consisted only from benzene rings, the second only from thiophene skeletons and the
third was composed from both of them. Besides, they contained on one side electron donating
group and on the other electron withdrawing substitution to create push-pull system. Strategy
of the synthesis with the yields of reactions are shown at the following Scheme 1 [4].
I
Br
BOHHO
Br
N
+
N
N
B
OH
OHO
O
(Ph3P)4Pd, K3PO4
PdCl2(dppf), K2CO3
86 %
86 %
S Br+
O
B
S
OOH
OH NBS
S
O
Br
B
OH
OH
N
PdCl2(dppf),K3PO4S
ON
75 % 96 %77 %
Pd(PPh3)3
K3PO4 ,
SB
OH
OH SBr O+
S
SO
PdCl2(dppf), K3PO4S
SO
NBS Br
SB
O
O
S
SO
S70 % 90 %70 %
Scheme 1.
Final step in all the cases included reaction of these three cyclic systems leading to
introduction of electrowithdrawing substitution. The reaction proceeded via aldehyde group
(Scheme 2) [5].
Scheme 2.
N N
C NOH
KOH
piperidine
70 %
60 %
O
+N
N
+
N
O
O
CH3OH
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Electrochemical measurements
Redox signals of 1mM solutions of measured compounds in dichloromethane with 0.1M
tetrabutylammonium hexafluorophosphate (TBAPF6) as the supporting electrolyte were
measured against the Ag/AgCl/3M KCl aqueous reference electrode separated from the main
electrolytic compartment by a fritted junction containing the same supporting electrolyte
(0.1M TBAPF6 in DCM). A platinum electrode (area 7.1 mm2) and a platinum wire were used
as the working electrode and the counter electrode, respectively, in conventional three-
electrode voltammetric arrangement. For the potential scaling, necessary for the determination
of HOMO and LUMO energies, ferrocene voltammetric response (0.1mM) in the same
medium was evaluated.
Spectral measurements
Absorption, emission and excitation spectra of 10-5
solutions of prepared compounds in DCM
were measured. Schimadzu UV-1601 Spectrophotometer and Spectrofluorimeter FLS 920,
Edinburgh were used.
Quantum calculations
Molecular geometries have been optimized at the B3LYP/6-31G* level. Mullikan charges
have been determined for the optimized geometries at the same level of theory. Orbital
energies have been determined from an additional B3LYP calculation employing the 6-311G*
basis set. All calculations have been performed on isolated molecules in the gas phase, i.e.
solvent effects have been neglected.
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3. RESULTS AND DISCUSSION
*calc. = DFT calculation, *CV = cyclic voltammetry, *opt. = absorption spektra, Eg = energy gap
They were synthesized 9 new compounds applying modern cross-coupling reactions and
afterwards were tested for their properties.
4. CONCLUSION
As the most interesting result, we can consider the strongly different abilities of the individual
aromatic systems to transfer electronegativity perturbations from the substituted nucleus
throughout the molecule. The least flexible system turns out to be the terphenyl unit, possibly
due to significant rotation angles (of ca. 30o) caused by the Pauli repulsions between hydrogen
atoms of neighboring benzene units. The most flexible molecule is, according to our
calculations, the system with alternating benzene and thiophene units, where the electron
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density perturbation is largest at the carbon bearing the diethylamine unit, i.e. at the molecular
end opposite to the substituted position.
We assume that this is caused by a joint effect of smaller rotation angle (ca. 20o) and an
interference of electronic perturbations in the alternating system. The rotation angle itself is
namely smallest for the system consisting of three thiophene nuclei (0o
for the aldehyde and
dicyano structures, 15o for the nitrone structure), causing however only intermediately
efficient electron transfer throughout the molecule.
We expect that an inclusion of solvent effects in the calculations, planned for our future work,
will improve the agreement of calculated HOMO-LUMO gaps with the cyclic voltammetry
experiment.
5. ACKNOWLEDGEMENT
This research has been supported by Project SIX CZ.1.05/2.1.00/03.0072. Authors also thank
to J. Krausko for help with molecular spectra measurement and their discussion during
evaluation.
6. REFERENCES
[1] Beaujuge P. M., Amb C. M., Reynolds J. R..: Acc. Chem. Res. 43 (2010), 1396-1407.
[2] Li C., Wonneberger H. : Adv. Mater. 24 (2012), 613-36.
[3] Kivala M., Diederich F.: Acc. Chem. Res. 42 (2009), 235-248.
[4] Martin A. R.,Yang Y.: Acta Chem. Scand. 47 (1993), 221-230.; Miyaura N., Suzuki A.: Chem. Rev. 95
(1995), 2457-2483.; Suzuki A. : J. Organomet. Chem. 576 (1999), 147-168.
[5] Buchlovič M., Man S., Kislitson K., Mathot Ch., Potáček M.: Tetrahedron 66 (2010), 1821-1826.
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DIAMOND COATED QUARTZ CRYSTAL MICROBALANCE SENSOR
FOR DETECTION OF PROTEIN ADSORPTION
Václav PROCHÁZKA1, 2*
, Pavel KULHA2, Tibor IŽÁK
1, Egor UKRAINTSEV
1, Marian
VARGA1, Alexander KROMKA
1
1 Institute of Physics, Czech Academy of Sciences, Cukrovarnicka 10, 162 00 Prague, Czech Republic
2 Department of Microelectronics, Faculty of Electrical Engineering, Czech Technical University, Technicka
2, 166 27 Prague, Czech Republic
Abstract
In this study, we present a sensor based on a bulk acoustic quartz crystal microbalance (QCM)
piezoelectric device coated with nanocrystalline diamond (NCD) as a sensitive layer for
detection of adsorbed proteins. Three kinds of proteins were tested (FBS, BSA, FN). The
higher frequency shift in serial resonance almost 650 Hz was observed for sensor with double
side FBS coating. Moreover, oxygen terminated surfaces exhibited higher shift in frequency
for single side covered QCM sensors, than the hydrogenated surfaces.
1. INTRODUCTION
It was already shown that QCM sensor is an attractive platform for biological studies
including detection of different bacteria [1,2] or cell events (e.g. metabolism and adhesion of
dying cells on gold [3]), etc. The detection principle is attributed to the change of the QCM
resonant frequency (serial). The most of QCM devices are using gold as the detection layer. In
comparison to gold, the diamond-coated QCMs should have additional advantages as
controllable and stable surface functionalization (e.g. surface treatment, covalent binding of
small molecules or even DNA etc.). Moreover, the cell-diamond interaction (adhesion,
proliferation, etc.) can be influenced by changing its morphology (porous film, nano-wires,
etc.).In this study, we fabricated and deployed diamond-coated QCMs for the detection of
adsorbed proteins as the first event before the cell cultivation studies.
2. MATERIALS AND METHODS
Quartz crystal microbalance devices were coated with continuous nanocrystalline diamond
layer (~300 nm thick) grown by pulsed linear antenna microwave plasma chemical vapour
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deposition system [4,5]. Afterwards, the diamond surfaces were hydrogen and oxygen-treated
by appropriate plasma procedure to obtain hydrophobic and hydrophilic character,
respectively.
Three kinds of proteins were used: fetal bovine serum (FBS), bovine serum albumine (BSA)
and fibronectin (FN), which are the most often serving for eukaryotic cells cultivation.
Proteins were applied on the QCM active area by the drop off technique. The drop of solution
was kept on the sample for 10 min, then the sample was rinsed by deionized water and dried
by air gas flow. This procedure led to the formation of ~3 nm thick protein layer [6]. We
compare QCM response with proteins dropped on its one or both sides, respectively.
To measure the serial resonant frequencies a frequency sweep measurement (with centre
frequency close to 10 MHz at first natural mode, and span of 40 kHz) was performed.
3. RESULTS AND DISCUSSION
The QCM sensors serial resonant frequencies were significantly influenced by the protein
adsorption. Shifts in serial resonant frequency were evaluated due to higher sensitivity and
stability [7] as graphically shown in Fig. 1 and summarized in Table 1.
Figure 1.: Impedance characteristics of oxygen-terminated sample O-4 without and with FBS solution coated on
single and double side (Fig. 1B. shows detailed view of the frequency shifts in serial resonance)
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Table 1: Measured frequency shifts of oxygen- and hydrogen-terminated QCM covered with different solutions
Sample/Solution O/FBS O/BSA O/FN H/FBS H/BSA H/FN
Frequency shift – single side (ΔHz)
-174 -219 -154 -128 -202 -50
Frequency shift – double side (ΔHz)
-419 -302 -282 -643 -318 -206
Thickness (nm) 2.8±1.1 1.8±0.4 3.0±1.0 2.8±0.6 2.4±0.3 2.4±0.5
The lower shift was observed for FN on H-terminated diamond, whereas the highest
frequency shift was observed for FBS applied on both sides of QCM. This effect can be
caused by different kinds of entrapment. Related to that, it seems, that frequency shifts are
independent to thickness of the protein layer. Oxygen terminated surfaces exhibits higher
sensitivity to fibronectin, these differences may be caused by different macromolecular
conformation of proteins on H-terminated diamond and O-terminated diamond as it was
described in [6]. For the single side coated QCM the highest shift was for BSA and lowest for
FN on both H/O-NCD sensors. However, the double side coated QCM have highest shift for
FBS, and lowest again for FN on both H/O NCD sensors.
4. CONCLUSION
Biosensors based on diamond-coated QCM were successfully fabricated and tested. Three
kinds of proteins were studied and each one exhibits obvious shift on resonant frequency. For
future research options can be considered use of other types of cultivation solutions as YPD,
or buffer solution (PBS) to examine its influence. In the next step, we plan to perform
measurements with the set of eucaryotic cells with adhesive and nonadhesive properties.
5. ACKNOWLEDGEMENT
The work has been supported by CTU grant no. SGS15/159/OHK3/2T/13 - Development of
Nanocrystalline Diamond Layers Based Biosensor for Measurement Properties of Living
Cells and by the Czech science foundation research project P108/12/G108 (VP, MV, AK).
6. REFERENCES
[1] Su, X. L., & Li, Y.: Biosensors and Bioelectronics, 21(6), (2005), 840–848.
[2] Strauss, J., Liu, Y., et all.: JOM, (2009), 71–74.
[3] Nowacki, L., Follet, J., Vayssade, et all.: Biosensors and Bioelectronics, 64, (2015), 469–476
XV. Workshop of Physical Chemists and Electrochemists
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[4] Izak, T., Babchenko, O., Varga, et all.: Physica status solidi (b), 249(12), (2015)., 2600-2603.
[5] Varga M., Laposa A., Kulha P., et all.: Key Engineering Materials, 605, (2014), 589-592
[6] Rezek, R., Krátká, M., Kromka, et all.: Biosens. Bioelectron. 26, (2014), 1307-1312.
[7] Bouřa, A., Kroutil, J.: ASDAM 2014 Conference Proceedings. Bratislava: Slovak University of
Technology in Bratislava, (2014), 217-220
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ELECTROCHEMICAL FABRICATION OF THIN NANOPOROUS
TITANIA SURFACES
Kateřina PŘIKRYLOVÁ1,2*
, Jana DRBOHLAVOVÁ1,2
1 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of
Technology, Technická 3058/10, 616 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technická 3058/10, 616 00 Brno,
Czech Republic
Abstract
Nanoporous titanium dioxide nanostructure surfaces on silicon wafers were prepared via one-
step and two-step anodic oxidation methods in organic electrolyte containing ammonium
fluoride. The topography of nanoporous surfaces were observed by scanning electron
microscopy (SEM) and related to the fabrication parameters, namely applied voltage and
length of anodization.
1. INTRODUCTION
The titanium dioxide is an important wide bandgap semiconductor in the field of
photocatalysis due to its strong oxidizing ability [1], superhydrophilicity, chemical stability,
long durability, nontoxicity, low cost and transparency to visible light. Self-ordered TiO2
nanostructured and nanoporous surfaces have great potential as a superior photocatalyst due
to their valuable high surface area. Also is not necessary to remove remains of nanoparticles
such as in TiO2 suspensions [2].
The TiO2 nanostructured and nanoporous surfaces can be fabricated by template method, sol-
gel, hydrothermal processes and anodic oxidation method. Anodization has become one of the
most popular method because of its high controllability, low cost, quickness and
reproducibility. Morphological structure of anodized TiO2 can be modified by changing the
preparation conditions like anodization time, applied voltage, temperature, and electrolyte
composition, in particular fluoride concentration, water and organic additives content, and pH.
[3,4]
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2. MATERIAL AND METHODS
Titanium (99.99%, Porexi, CZ), ethylene glycol p.a. (C2H6O2, PENTA, CZ) and ammonium
fluoride p.a. (NH4F, Reidel-de Haen) were used as purchased. Deionized water (18.2 M )
was obtained from Millipore RG system MilliQ (Millipore Corp., USA).
The titanium layer with thickness of 1 µm was sputter-deposited on n-dopped silicon wafer
covered with SiO2 layer with thickness of 995 nm. Nanoporous TiO2 surfaces were fabricated
either by one-step or by two-step anodization approach in electrolyte containing ethylene
glycol, 0.3 wt% NH4F and 2 vol% H2O. [5] One step anodization was performed at pH 7, 60
V and room temperature. In the case of two-step anodization, the constant potential of 60 V
was applied in the first step, while it varied from 40 to 100 V in the second step. [6]
3. RESULTS AND DISCUSSION
Figure 1. represents the SEM images of anodic TiO2 nanoporous film fabricated in organic
electrolyte at 60 V. The pore diameter was about 60 nm and the length of anodized titania was
about 2 µm.
Figure 1.: SEM images of anodic TiO2 nanoporous structure fabricated by one-step anodization, top view (left)
and cross-section view (right).
Surface morphology of TiO2 nanoporous layer prepared via two-step anodization is shown in
Figure 2. The thin titania upper layer created in the first short anodization was carefully
removed by ultrasonic method and then many nanodimples were revealed in the titanium
layer below. These nanodimples with uniform size of 60 nm are hexagonally distributed over
the surface. During the second anodization the upper oxide layer was partly dissolved due to
the aggression of fluoride anions from the electrolyte, which resulted in formation of
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nanoporous structure. The nanostructures length depends on the voltage applied during the
second step of anodization.
Figure 2.: SEM images of anodic TiO2 nanoporous surface fabricated by two-step anodization, top view (left)
and cross-section view (right).
4. CONCLUSION
The present research demonstrates a method for the fabrication of anodic TiO2 surfaces in
organic electrolyte containing NH4F via one-step and two-step anodization method. SEM
images showed the anodized titania surfaces with nanoporous character. TiO2 nanoporous
layer prepared via two-step anodization has unique upper structure, which can serve as a
protective layer for lower nanostructures or as a stable matrix for supporting nanoparticles.
This TiO2 nanoporous layer can be further doped with noble metals (eg. silver, gold) in order
to improve the photocatalytical efficiency in VIS region.
5. ACKNOWLEDGEMENT
The research was supported by project no. FEKT-S-14-2300 A new types of electronic
circuits and sensors for specific applications.
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6. REFERENCES
[1] Tsuji E. et al.: Applied Surface Science, 301(2014), 500-507.
[2] Wang W. Y. and Chen B. R.: International Journal of Photoenergy, (2013), 12.
[3] Nischk M. et al.: Applied Catalysis B-Environmental, 144 (2014), 674-685.
[4] Erjavec B. et al.: Catalysis Today, 241 (2015), 15-24.
[5] Chen B. et al.: Rsc Advances, 4 (2014), 29443-29449.
[6] Zhang Z. a Wu H.: Chemical Communications, 50 (2014), 14179-14182.
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VOLTAMMETRY OF GUANOSINE AND GUANOSINE
MONOPHOSPHATE ON A PENCIL GRAPHITE ELECTRODE
Mehdi RAVANDEH, Libuse TRNKOVA*
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech
Republic
Abstract
Guanosine (Guo) and guanosine monophosphate (GMP) are noted for their biological effects
in human system. In this study, the voltammetry of Guo and GMP on pencil graphite
electrode (PeGE) was studied. In the first voltammetric cycle, oxidation peaks at ca. 1.1 V vs.
Ag/AgCl were observed for both compounds. In the second cycle at about 0.6 V for Guo and
GMP an additional oxidation peak was found. The further electrochemical experiments
indicated that the new peak corresponds to 8-oxoguanine moiety which was after the
oxidation process of Guo or GMP adsorbed on PeGE surface. The intensity of 8-oxoguanine
peak for Guo was higher than for the GMP, that is probably due to the effect of the phosphate
group of GMP. The effect of pH on both the first and the second cycle is manifested by
negative shift of oxidation signals with increasing of pH. In addition, the voltammograms of
Guo and GMP were analyzed using the elimination function E4, indicating the adsorption
processes for both Guo and GMP by the signal in a peak-counterpeak form.
1. INTRODUCTION
Purine nucleosides are noted for their biological effects in human system. Their detection and
determination has become increasingly important in the field of biomedical research [1].
Among of purine nucleosides, guanosine (Guo) is a naturally endogenous compound with a
wide spectrum of biological activities. Guo stimulates neurotrophic factor synthesis, which
protects neurons from excitotoxic death and mediates the process of RNA splicing [2].
Guanosine-5´-monophosphate (GMP) is one of the derivatives of guanosine in RNA. GMP
concentration plays a crucial role in many functions related to normal cellular metabolism,
blood pressure and cardiac activities [3]. In order to study the function of Guo and GMP in
the organism, determination of those are gaining a lot of interest in different fields including
biology, medicine and pharmacy [4]. Electrochemical technique is a powerful method for
detection of Guo and GMP due to the advantages of rapid response, low cost, simple
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operation, time effectiveness, high sensitivity, but in most cases it suffers from poor
selectivity [5]. Many mathematical models have been proposed to extract useful information
from the CV data and elimination voltammetry is one from such models. Elimination
voltammetry with linear scan (EVLS), an electrochemical method comprising the elimination
of some particular currents from the measurements of linear scan voltammetry was first
proposed by Dracka and simultaneously verified by Trnkova [6]. In this study, the data from
the first and second cyclic voltammetry cycles of Guo and GMP were investigated by the
elimination voltammetry procedure (EVP).
2. MATERIALS AND METHODS
Chemicals
Guanosine (Guo) and guanosine-5´-monophosphate (GMP) were purchased from Sigma
Chemical Co. (St. Louis, U.S.A.) and both the compounds were used as received without
further purification. All other reagents were of analytical grade and were used as received.
Linear Sweep voltammetry (LSV)
Linear sweep voltammetry was carried out on an AUTOLAB analyzer (Metrohm, Ecochemie,
Netherlands) connected with a VA-Stand 663 (Metrohm, Zurich, Switzerland) and software
Nova. A standard cell with three electrodes was used. The working electrode was a pencil
graphite electrode (PeGE) (Tombow 05 HB, Japan). The electrode Ag/AgCl/KCl (3 M) as a
reference electrode and platinum wire as an auxiliary electrode were used.
3. RESULTS AND DISCUSSION
The EVP, as an unconventional electrochemical method capable of eliminating or conserving
the selected particular currents (diffusion, charging, and kinetic currents) from the total
voltammetric currents measured at three scan rates, provides more sensitive signals compared
to LSV. From the point of view of increasing current sensitivity, resolution capability and
indication of adsorption, the EVLS procedure eliminating the capacitance and kinetic
currents and conserving the diffusion current component was chosen [7]. This EVLS function,
depicted as function E4, was expressed as: f ( I ) = −11.657 I1/2 + 17.485 I −5.8284 I2 ,
where I1/2, I, and I2 are total voltammetric currents measured at different scan rates. In the
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case of totally adsorbed electroactive species the course of this E4 function corresponds to the
peak-counterpeak form (Fig. 1).
Figure 1: Linear sweep and elimination voltammograms of first and second cycles for Guo and GMP (pH 3.9)
As one can see in Fig. 1 the EVP shows an adsorption process for Guo and GMP in both the
first and second cycle, and also a new peak at the second cycle in the potential 0.6 V for Guo
and GMP appears. Further electrochemical experiment indicated that the new peak
corresponds to 8-oxoguanine moiety. However, the peak of 8-oxoguanosine for Guo in
second cycle is higher than the GMP peak - that is probably due to phosphate group of GMP.
As a result, PeGE shown very good sensibility for detection of Guo, GMP and also 8-
oxoguanine moiety.
4. CONCLUSION
The effect of first and second cycles of Guo and GMP on pencil graphite electrode was
investigated by elimination voltammetry in phosphate-acetate buffer solution (pH 3.9).
Elimination voltammetry procedure increased the peak currents and also revealed an
adsorption process at the surface of electrode as indicated by the peak-counterpeak shape. In
the second cycle, a new peak appeared corresponding to 8-oxoguanosine or 8-oxoGMP
moieties. The results of determination of GMP and Guo and also 8-oxoguanine moiety
showed that the PeGE is very good and inexpensive electrode for the determination of RNA
and DNA bases. The study of electrochemical oxidation of guanine to 8-oxoguanine is very
important due to a frequent transformation of guanine to 8-oxo-7,8-dihydroguanine (8-
-30
20
70
120
170
220
0.2 0.4 0.6 0.8 1 1.2
E4 o
r I (
µA
)
E (V vs. Ag/AgCl)
Guo,E4,C1,C2,pH3.9
400mv/s,C1 E4,C1 400mv/s,C2 E4,C2
-110
-60
-10
40
90
140
190
240
290
0.2 0.4 0.6 0.8 1 1.2
E4 o
r I (
µA
)E (V vs. Ag/AgCl)
GMP,E4,C1,C2,pH3.9
400mv/s,C1 E4,C1 400mv/s,C2 E4,C2
8-oxoGua
Guo
8-oxoGua
GMP
-10
0
10
20
30
40
0.5 0.7
E4 o
r I (
µA
)
E (V vs. Ag/AgCl)
-10
-5
0
5
10
0.5 0.7
E4 o
r I (
µA
)
E (V vs. Ag/AgCl)
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oxoGua) in DNA that occurs in cells as a result of the metabolic generation of reactive
oxygen species or exposure to agents that induce oxidative stress.
5. ACKNOWLEDGMENT
This research was supported by the project SIX CZ.1.05/2.1.00/03.0072 and by the projects:
MUNI/A/1452/2014 and LH 13053 KONTAKT II of the Ministry of Education, Youth and
Sports of the Czech Republic.
6. REFERENCES
[1] Goyal R. N., Gupta V. K., Oyama M., Bachheti N.: Talanta 71 (2007), 1110–1117.
[2] Yin H., Zhou Y., Ma Q., Ai S., Chen Q., Zhu L.: Talanta 82 (2010), 1193–1199.
[3] Sun W., Xu L., Liu J., Wang X., Hu S., Xiang J.: Croatica Chemica Acta 86 (2013), 129–135.
[4] Jeevagan A.J., John S.A.: Electrochimica Acta 95 (2013), 246–250.
[5] Zagal J.H., Griveau S., Silva J.F., Nyokong T., Bedioui F.: Coordination Chemistry Reviews 254
(2010), 2755–2791.
[6] Bhatt S., Trivedi B.: International Journal of Electrochemistry 2013 (2013), e678013.
[7] Navratil R., Pilarova I., Jelen F., Trnkova L.: International Journal of Electrochemical Science 8
(2013), 4397–4408.
[8] Cadet J., Douki T., Gasparutto D., Ravanat J. L.: Mutation Research 531(2003), 5–23.
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MODIFICATION OF CQDS MONITORED BY BRDICKA REACTION
Lukas RICHTERA1,2
, Vedran MILOSAVLJEVIC1,2
, Pavel KOPEL1,2
, David HYNEK1 and
Rene KIZEK1,2*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
One of possible alternatives of quantum dots (QDs) or their conjugates detection is
electrochemical detection. This paper presents the detection and characterization of carbon
quantum dots (CQDs) modified with polyethylene glycol and polyvinylpyrrolidone using
differential pulse voltammetry (Brdicka reaction). From data obtained it is obvious that this
electrochemical method can serve for qualitative and quantitative detection of modified
CQDs.
1. INTRODUCTION
QDs are nanoparticles with dimensions of the order of nanometers, containing several
hundred up to tens of thousands of atoms. For the purpose of stabilization and also because of
better interaction with biomolecules, surface modification of QDs is performed by polymers
[1]. QDs based on metals and metalloids may exhibit toxicity, so some none toxic alternatives
like CQDs are attentively investigated.[2] Considering to QDs fluorescence properties
fluorescence microscopy is logically most preferred method for detecting fluorescent QDs in
the case of in vivo and in vitro analysis and imaging. An alternative method of detection of
QDs and their conjugates is their detection by electrochemical methods.[3, 4]
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2. MATERIAL AND METHODS
C-dots synthesis
General method for the preparation of water soluble CQDs were adopted according to Wang
et al. [5]. Into a 100 ml three-neck flask were added 10 ml of ethylene glycol, 1.0 g of
polymer solution (polyethylene glycol – PEG, Mw ~8 kD, or polyvinylpyrrolidone - PVP, Mw
~10 kD) and 1.0 g of citric acid. The solution was heated (on 180 ºC for PEG, on 120 °C for
PVP) under flow of nitrogen (time of heaiting: 4 hours for PEG, 24 hours for PVP), and then
cooled down to ambient temperature. In to cooled solution water was added and then the
mixture was stirred for few minutes. Solution was subsequently dialyzed. Both samples were
than centrifugated at 10 000 RPM and the supernatants were used in experiments.
Electrochemical determination
Detection of CQDs by differential pulse voltammetry (DPV) was performed with 663 VA
Computrace instrument (Metrohm, Switzerland), using a standard cell with three electrodes.
A hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm2 was employed as the
working electrode. An Ag/AgCl/3M KCl electrode was used as the reference and carbon
electrode served as auxiliary. The Brdicka supporting electrolyte containing 1 mM
[Co(NH3)6]Cl3 and 1 M ammonia buffer (NH3(aq) + NH4Cl, pH = 9.6) was used. The
parameters of the measurement by differential pulse voltammetry were as follows: initial
potential of -0.55 V, end potential -1.80 V, deoxygenating with argon 15 s, deposition 0 s,
time interval 0.8 s, step potential 4.95 mV, modulation amplitude 25.05 mV, modulation time
0.03 s, scan rate 0.006187 V·s-1
. All measurements were carried out on air at 6.0±0.1°C.
3. RESULTS AND DISCUSSION
A study was performed on the effect of deposition time to the signal height. Individual
voltammograms with unresolved peaks were evaluated at particular values of potential (for
PEG and PEG modified CQDs: -0.61 V, -0.70 V and -0.90 V, and for PVP and PVP modified
CQDs: -0,57 V, -0,65 V and -0,79 V). The best results were obtained without deposition time
application. With increasing concentration of analyte the increase of intensity occurs in
limited range only. The situation is in all cases complicated by shift of unresolved peaks, as
well shown in Fig. 1. Low concentrations of analyte even do not lead to the expected response
in the intensity of the monitored signals. Instead, there is a significant shift signal. This shift
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can be explained by formation of a cobalt complex with a modifying material, i.e. PEG or
PVP, on the surface of the carbon quantum dots. In this interaction may participate hydroxyl
functional groups that terminate the individual chain of PEG, and the oxygen bridging group
in the case of PEG. In the case of PVP there is even another possibility of donor-acceptor
coordination via nitrogen atom. Consequently, after saturation of binding possibilities of
Co(II), the expected response of signal intensity increase can be observed.
Figure 1.: Differential pulse voltammetry of polymers and CQDs: (A) Voltammogram of pure PEG solution;
(B) Voltammogram of CQDs covered by PEG; (C) Voltammogram of pure PVP solution; (B) Voltammogram of
pure CQDs covered by PVP.
4. CONCLUSION
Direct quantitative evaluation of voltamograms obtained is due to the broad and overlapping
signals only partially possible. Mathematical separation of signals would allow to trace their
precise positions and highs and thus lead to the more accurate quantitative data evaluation.
5. ACKNOWLEDGEMENT
The financial support from STRATO-NANOBIOLAB (CZ/FMP.17A/0436) is highly
acknowledged.
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6. REFERENCES
[1] Algar WR, Tavares AJ, Krull UJ: Analytica Chimica Acta, 673 (2010), 1, 1-25
[2] Yang S-T, Wang X, Wang H, et all: The Journal of Physical Chemistry C, 113 (2009), 42, 18110-18114
[3] Pinwattana K, Wang J, Lin C-T, et all: Biosensors and Bioelectronics, 26 (2010), 3, 1109-1113
[4] Krejcova L, Hynek D, Kopel P, et all: International Journal of Electrochemical Science, 8 (2013), 4, 4457-
4471
[5] Wang F, Pang SP, Wang L, et all: Chemistry of Materials, 22 (2010), 4528-4530
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PRION PROTEIN AND ITS INTERACTIONS WITH METALS AND
METALLOTHIONEIN 3
Branislav RUTTKAY-NEDECKY1,2
, Eliska SEDLACKOVA1, Dagmar CHUDOBOVA
1,2,
Kristyna CIHALOVA1,2
, Vojtech ADAM1,2
and Rene KIZEK1,2*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
Total metallothionein content in bacterial cells expressing hPrPC or MT-3 proteins were
determined using differential pulse voltammetry (DPV). From experimental results it can be
concluded that hPrPC helps maintain the equilibrium level of metal ions in the cell and
protects it against oxidative stress caused by metal ions to a greater degree than the MT-3.
1. INTRODUCTION
Prions are self-replicating protein aggregates that play a primary role in a number of
neurological disorders in mammals. Prion protein (PrP) undergoes conformational
transformation that leads to the protein aggregation and its transition to the infectious cellular
pathogen [1]. A large number of studies suggests that the major role in prion diseases plays
conformational conversion of PrPC (the normal cellular prion protein) to PrP
Sc (its abnormal
isoform), which becomes infectious [2]. PrPC is a glycoprotein that interacts with many
divalent metal ions, particularly Cu2+
and Zn2+
[3]. Another protein having the ability to bind
metals, is also metallothionein (MT), which consists of several specific forms [4]. Regarding
formation of neurodegenerative diseases is significant metallothionein isoform MT-3 occuring
in the brain. One of the MT-3 functions in the brain is its participation in maintaining of the
optimal concentration of metal ions [5]. The aim of this study was the monitoring of heavy
metal ions influence on total MT content in E. coli bacterial cells expressing the PrPC or MT-3
protein. Any electrochemical determination was compared to a standard E. coli BL21 strain.
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2. MATERIAL AND METHODS
Preparation of samples
Sample (control strains – E. coli, E. coli – MT-3, E. coli - hPrPC or strains with addition of 10,
150 and 300 µM concentrations of copper, cadmium or zinc ions) was centrifuged by 8000
rpm for 10 minutes. To the pellet the liquid nitrogen was added. After evaporation, 1 ml of
phosphate buffer (pH 7) was added and samples were mixed for 30 minutes. 2 minutes of
ultrasound were used for the lysis of cells. After centrifugation by 8000 rpm for 10 minutes
the supernatant will be used in the following experiments. Further experimental details are
described in previous article [6].
Electrochemical measurement of metallothionein by differential pulse voltammetry
Differential pulse voltammetric measurements were performed with 747 VA Stand instrument
connected to 693 VA Processor and 695 Autosampler (Metrohm, Switzerland), using a
standard cell with three electrodes and cooled sample holder and measurement cell to 4 °C
(Julabo F25, JulaboDE). A hanging mercury drop electrode (HMDE) with a drop area of 0.4
mm2 was the working electrode. An Ag/AgCl/3M KCl electrode was the reference and
platinum electrode was auxiliary. The analysed samples were deoxygenated prior to
measurements by purging with argon (99.999 %) saturated with water for 120 s. Brdicka
supporting electrolyte containing 1mM Co(NH3)6Cl3 and 1M ammonia buffer (NH3(aq) +
NH4Cl, pH = 9.6) was used. The supporting electrolyte was exchanged after each analysis.
The parameters of the measurement were as follows: initial potential of -0.70 V, end potential
of -1.75 V, modulation time 0.057 s, time interval 0.2 s, step potential 0.002 V, modulation
amplitude -0.250 V, Eads = 0 V, volume of injected sample: 20 µl, volume of measurement
cell 2 ml (20 μl of sample and 1980 l Brdicka solution) for calibration curves. The volume
for the measurement of bacterial culture of E. coli and E. coli with MT-3 or hPrPC with metals
was 100 l of bacterial culture and 1900 l of Brdicka solution.
3. RESULTS AND DISCUSSION
Total content of metallothionein in the bacterial cells was determined using differential pulse
voltammetry (DPV). As samples control E.coli cells and E.coli cells expressing hPrPC or MT-
3 proteins were used. These cultures were exposed to two essential metal ions (Cu2+
, Zn2+
)
and one toxic metal ion (Cd2+
) (Fig. 1).
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Figure 1.: Metallothionein content in E. coli cells BL21 transformed with an empty plasmid, the plasmid
containing hPrPC or MT-3 gene after incubation with different concentrations of metals (25, 75, and 125 μM)
Cu (A), Zn (B) and Cd (C) compared to control cells without addition of metal.
Bacterial cells which had expressed hPrPC were better protected against oxidative stress
caused by the presence of metal ions. hPrPC together with a bacterial metallothionein act as
trap of metal ions and it also reduced levels of metallothionein at lower metal concentrations
compared to control.
4. CONCLUSION
The results of this work have contributed to the overall mosaic of scientific knowledge about
prion proteins and will be followed by further research in this area, which will help to
elucidate the function of these interesting proteins.
5. ACKNOWLEDGEMENT
Financial support from CEITEC CZ.1.05/1.1.00/02.0068 is highly acknowledged.
6. REFERENCES
[1] Prusiner S B, Scott M R, DeArmond S J, et al., Cell, 93 (1998), 337-348.
[2] Ortega-Cubero S, Luquin M R, Dominguez I, et al., Neurologia, 28 (2013), 299-308.
[3] Choi C J, Kanthasamy A, Anantharam V, et al., Neurotoxicology, 27 (2006), 777-787.
[4] Atrian S, Capdevila M, Biomol Concepts, 4 (2013), 143-160.
[5] Hozumi I, Asanuma M, Yamada M, et al., Journal of Health Science-Tokyo, 50 (2004), 323-331.
[6] Adam V, Chudobova D, Tmejova K, et al., Electrochimica Acta, 140 (2014), 11-19.
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MICROFLUIDIC CHIP WITH AMPEROMETRIC DETECTION FOR
MONOSACCHARIDES DETERMINATION
Jan SLAVÍK1, Jaromír HUBÁLEK
1*
1 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
This paper presents a microfluidic system integrated into the chip for separation of
monosaccharides. The chip consists of polydimethylsiloxane (PDMS) and glass (Pyrex) with
integrated gold electrodes for amperometric detection. The chip was tested by the separation
of glucose and fructose.
1. INTRODUCTION
One of the current trends in analytical techniques is there miniaturization. Miniaturization
reduce analysis time and also reduces the amount of consumed materials. Microfluidic chips
have become an effective tool for the analysis of samples. New materials such as PDMS open
up new possibilities for fabrications of chips and possibilities of detection.
2. MATERIAL AND METHODS
Microfluidic chip is consists of upper and lower substrate. Fabrication of the upper substrate
shows figure 1A. Fabrication include classical micro-manufacturing fabrications processes of
metal deposition, spin coating, etching and cutting of components of PDMS 1:10 (curing
agent:polymer). After 1.5 hours at 90 °C PDMS was cured and peeled off from the silicon.
Upper substrate formed microchannels with 50 µm thickness. Fabrication of the lower
substrate shows figure 1B. The lower substrate formed gold electrodes with 100 nm thickness.
The lower substrate was activated by oxygen plasma (200 W, 60 seconds) and then it was
pressed against the upper substrate, thereby the two substrates was fixed together. The
resulting compilation shows figure 2.
During fabrication must be taken in account warming of electrolyte. The current through the
electrolyte should not exceed 100 nA, otherwise electrolyte is too hot and it creates bubbles in
it. The current flowing through the electrolyte could be affected by separation voltage,
concentration of electrolyte and the surface profile of a microchannel.
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Figure 1.: A. Fabrication process of the upper substrate of PDMS with microchannels; B. fabrication procecess
of lovwer substrate with integrated electrodes
Figure 2.: Scheme of microfluidic chip; A - upper substrate (PDMS); B - lower substrate (Pyrex with integrated
electrodes); C - detail of detection area
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3. RESULTS AND DISCUSSION
Microchannels were filled with 10 mM NaOH by vacuum. The reservoir 1 was filled with 10
mM NaOH, 5 mM glucose and 5 mM fructose. This solution was drain into the reservoir 2 by
vacuum to fill crossing microchannels. Then was applied separation voltage 1000 V (250
V/cm) between reservoirs 3 and 4. Electroosmotic flow tear all liquid flow under electric field
towards the cathode (reservoir 4). Different charge of monosaccharides causes different speed
of their migration. The monosaccharides were detected amperometrically at the exit of
microchannel at a potential of 0.4 V. The result of the separation shows figure 3.
Figure 3.: Separation of 5 mM fructose and 5 mM glucose in 10 mM NaOH
4. CONCLUSION
Paper presented fabrication processes for the microfluidic chip with amperometric detection
of monosaccharides. The chip was successfully tested. The paper also presented problems,
which must be taken into account for optimization of manufacturing processes and detection
method.
5. ACKNOWLEDGEMENT
The work has been supported by project STI-S-14-2523.
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6. REFERENCES
[1] O’Shea T, Lunte S: Current Separations, 14 (1995), 1, 18-23
[2] Engstrom-Silverman Ch, EWING A: Journal of Microcolum Separations, 3 (1991), 2, 141-145
[3] Vanderveer IV R, et. All.: Electrophoresis 25 (2004), 3528-3549
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VOLTAMMETRIC DETERMINATION OF BICARBONATE
Filip SMRČKA1*
, Jakub VANĚK1,2
, Přemysl LUBAL1,2
1 Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
2 Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Abstract
Electrochemical properties of LnIII
complexes with H2DO2A (1,4,7,10-
tetraazacyclododecane-1,7-diacetic acid) and H3DO3A (1,4,7,10-tetraazacyclododecane-
1,4,7-triacetic acid) make them perfect candidates for use in many chemical, biological and
environmental systems. H2DO2A and H3DO3A are hexa- and heptadentate ligands forming
very stable binary complexes with europium(III) ion, where three, resp. two coordination sites
are occupied by water molecules. These complexes form ternary complexes with small tri-
and bidentate ligands, e.g. carbonate, oxalate, phosphate, etc. Different stability constants of
those ternary complexes can be utilized for electrochemical selective determination of anions.
1. INTRODUCTION
Ln(III) complexes with macrocyclic ligands (mainly DOTA derivatives) are commonly used
as radiopharmaceuticals (90
Y, 153
Sm, 166
Ho, 177
Lu) or MRI contrast agents (Gd) in medicine or
as luminescent probes (Eu, Tb in VIS and Yb, Nd in NIR regions). The H2DO2A and
H3DO3A are hexa- and heptadentate macrocyclic ligands that yield very stable complexes
with europium(III) ion. Both complexes also form ternary lanthanide(III)-containing species
with both bi- and tridentate ligands. The binary complexes of the Ln(III)-H2DO2A and
Ln(III)-H3DO3A may be employed for determination of anions known to form the ternary
complexes [1]. In this contribution, we demonstrate selective anionic sensors suitable for
carbonate anion determination based on the [Ln(H2O)2(DO2A)] and [Ln(H2O)2(DO3A)]
complexes. The results shown here suggest a potential utility of this sensor for a construction
of sensor arrays.
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Figure 1: Scheme of [Eu(DO2A)(dipicolinate)]- and [Eu(DO3A)(picolinate)]
- ternary complexes.
2. MATERIAL AND METHODS
Cyclic voltammetry measurements were performed on Metrohm 910 PSTAT mini
(Switzerland) using Screen Printed Electrodes (SPEs) (Figure 2).
Figure 2. Metrohm 910 PSTAT mini equipment employed for CV analysis
3. RESULTS AND DISCUSSION
The formation of ternary Eu(III) species was followed by cyclic voltammetry. As it can be
seen on example of [Eu(DO3A)(picolinate)]- or [Eu(DO2A)(dipicolinate)]
- (Figure 1), the
formation of ternary Eu(III) complex is accompanied by increase of significant change of
electrochemical signal (Figure 3). Adding bicarbonate anion in solution, the new more stable
ternary [Eu(DO3A)(Carb)]2-
or [Eu(DO2A)(Carb)]2-
complex is formed which led to original
record (Figure 4).
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Figure 3: The change of electrochemical signal as consequence of the formation of [Eu(DPA)(DO2A)]
ternary complex
Figure 4: The change of electrochemical signal as consequence of the formation of [Eu(DO2A)(Carb)]
ternary complex
4. CONCLUSION
The bound water molecules in the [Eu(H2O)2(DO3A)]/[Eu(H2O)3(DO2A)] complex
undergoes substitution with various anions to form stable ternary adducts. The proposed
analytical procedure using the ternary complexes [Eu(DO3A)(L)]/[Eu(DO2A)(L)] (L =
picolinate, dipicolinate) can be used for a fast selective and sensitive determination of
carbonate/bicarbonate in the milimolar concentration range in biological and water samples.
5. ACKNOWLEDGEMENT
This work was supported by Ministry of Education of the Czech Republic (ME09065), Grant
Agency of Czech Republic (grants 13-08336S) and EU (CEITEC CZ.1.05/1.1.0/02.0068)
program.
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
-50
-40
-30
-20
-10
0
10
20
I/A
E/V
DPA added
-1.0 -0.8 -0.6 -0.4 -0.2 0.0-50
-40
-30
-20
-10
0
10
20
I/A
E/V
Carbonate added
[Eu(H2O)3(DO2A)] + DPA [Eu(DPA)(DO2A)] + 3 H2O
[Eu(DPA)(DO2A)] + Carb [Eu(DO2A)(Carb)] + DPA
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6. REFERENCES
[1] Vaněk J., Lubal P., Hermann P., Anzenbacher P. Jr.: J. Fluorescence 23 (2013), 57-69.
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NADPH- AND NADH-DEPENDENT OXIDATION OF DNA ADDUCT
FORMATION BY BENZO[A]PYRENE CATALYZED WITH HUMAN
CYTOCHROME P450 1A1
Marie STIBOROVÁ1*
, Radek INDRA1, Michaela MOSEROVÁ
1, Petr HODEK
1, Miroslav
ŠULC1, Heinz H. SCHMEISER
2, Eva FREI
2, Volker M. ARLT
3
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech
Republic
2 German Cancer Research Center (DKFZ), Heidelberg, Germany
3 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health,
King’s College London, United Kingdom
Abstract
Oxidation of and DNA adduct formation by benzo[a]pyrene (BaP) by human cytochrome
P450 (CYP) 1A1 enzyme are catalyzed not only by the NADPH:CYP reductase-mediated
mechanism, but can also be mediated by the system composed of this human CYP,
cytochrome b5, NADH:cytochrome b5 and NADH.
1. INTRODUCTION
Benzo[a]pyrene (BaP) is a genotoxic carcinogen that covalently binds to DNA after metabolic
activation by cytochrome P450 (CYP) [1,2]. CYP1A1 is the most important enzyme in BaP
bioactivation [2,3], in combination with microsomal epoxide hydrolase (mEH). First,
CYP1A1 oxidizes BaP to an epoxide that is then converted to a dihydrodiol by mEH (i.e.
BaP-7,8-dihydrodiol); then further bio-activation by CYP1A1 leads to the ultimately reactive
species, BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) that can react with DNA, forming
preferentially the 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]py-
rene adduct [4]. BaP is, however, oxidized also to other metabolites such as the other
dihydrodiols, BaP-diones and hydroxylated metabolites. Even though most of these
metabolites are the detoxification products, BaP-9-ol is a precursor of 9-hydroxy-BaP-4,5-
epoxide, which can form another adduct with deoxyguanosine in DNA [5,6]. Therefore,
regulation of CYP1A1-mediated oxidation of BaP leading to either metabolites forming
BPDE, 9-hydroxy-BaP-4,5-epoxide or the BaP metabolites that are the detoxification
products is of major importance
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However, there are still not clearly explained how an electron transfer on CYP1A1 during
BaP oxidation by the microsomal mixed-function-oxidase (MFO) enzymatic system occurs.
Generally, NADPH:CYP reductase (POR) was considered to be the essential reductase
transferring the electrons from NADPH to this CYP during its reaction cycle [6]. However,
cytochrome b5, an additional component of the MFO system might influence this electron
transfer. Whereas POR is considered as an essential constituent of the electron transport chain
towards CYP, the role of cytochrome b5 is still quite enigmatic. Likewise, a potential of
NADH as a donor of electrons to the CYP-mediated reaction cycle is still not exactly known.
Even though the second electron in the CYP reaction cycle might also be provided by the
system of NADH:cytochrome b5 reductase, cytochrome b5 and NADH, there is still rather
enigmatic whether this system might participate in donation of the first electron to CYP.
Therefore, here we investigated the effect of cytochrome b5, its reductase, NADH:cytochrome
b5 reductase and NADH on a potency of human CYP1A1 to oxidize BaP to its metabolites
both detoxifying this carcinogen and activating it to BaP-DNA adducts in vitro.
2. MATERIAL AND METHODS
Microsomes isolated from insect cells transfected with baculovirus constructs containing
cDNA of human CYP1A1 and expressing POR (Supersomes ), and human recombinant
CYP1A1 reconstituted with other components of the microsomal mixed function oxidase
system in liposomes were used as model enzyme systems. HPLC was used to separate and
identify BaP metabolites [7] and the 32
P-postlabling method to detect and quantify BaP-
derived DNA adducts [6].
3. RESULTS AND DISCUSSION
Human CYP1A1 expressed in Supersomes , in which human POR is also expressed,
metabolized BaP in the presence of both NADPH and NADH to up to eight metabolites (BaP-
9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione,
BaP-3-ol, BaP-9-ol and a metabolite with unknown structure) and activated this carcinogen to
form two BaP-DNA adducts. One of them was identified as 10-(deoxyguanosin-N2-yl)-7,8,9-
trihydroxy-7,8,9,10-tetrahydro-BaP (dG-N2-BPDE, adduct 2) and another adduct, derived
from reaction of 9-hydroxy-BaP-4,5-epoxide with guanine in DNA (adduct 1) [5,6], was also
formed. Levels of BaP metabolites and BaP-DNA adducts were analogous when the reaction
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was mediated either by NADPH or by NADH. Addition of cytochrome b5 to this CYP1A1
system led to an increase in BaP oxidation and levels of DNA adducts.
Human CYP1A1 reconstituted with POR and/or cytochrome b5 in liposomes oxidized BaP in
the presence of NADPH only to five metabolites (BaP-1,6-dione, BaP-3,6-dione, BaP-3-ol,
BaP-9-ol and a metabolite with unknown structure), and formed only the DNA adduct 1. The
CYP1A1 in this reconstitution system was, however, ineffective in the presence of NADH; no
BaP metabolites and BaP-DNA adducts were formed when NADH was used as a cofactor.
Human CYP1A1 reconstituted with NADH:cytochrome b5, cytochrome b5 and NADH in
liposomes generated these BaP metabolites and the BaP-DNA adduct 1, too, while this system
without cytochrome b5 was ineffective. Addition of microsomal epoxide hydrolase (mEH) to
the in-vitro incubations containing BaP, CYP1A1 reconstituted either with POR and NADPH
or NADH:cytochrome b5, cytochrome b5 and NADH resulted in formation of all BaP
metabolites found in SupersomesTM
, and also in generation of the DNA adduct (dG-N2-
BPDE).
4. CONCLUSION
The results found in this work demonstrate for the first time that the enzymatic system
consisting of human CYP1A1, NADH, NADH:cytochrome b5 reductase and cytochrome b5 is
capable of oxidizing BaP to its metabolites both detoxifying this carcinogen and forming BaP-
DNA adducts in vitro. They also indicate that NADH in the system of NADH:cytochrome b5
reductase and cytochrome b5 can act as a sole electron donor both for the first and second
reduction of CYP1A1 in its reaction cycle during metabolism of BaP in vitro.
5. ACKNOWLEDGEMENT
The work has been supported by GACR (15-02328S) and Charles University (UNCE
204025/2012)
6. REFERENCES
[1] IARC: IARC Monographs of Evaluation of Carcinogens. Risk of Chemicals for Human, 92 (2010), 1-853
[2] Baird WM, Hooven LA, Mahadevan B: Environmental and Molecular Mutagenesis, 45 (2005), 106–114
[3] Hamouchene H, Arlt VM, Giddings I, et al.: BMC Genomics, 12 (2011), 333
[4] Phillips DH, Venitt S: International Journal of Cancer, 131 (2012), 2733-2753
XV. Workshop of Physical Chemists and Electrochemists
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[5] Fang A.H., Smith W.A., Vouros P., et al.: Biochemical and Biophysical Research Communication, 281
(2001), 383-389
[6] Stiborová M., Moserová M., Cerná V., et al.: Toxicology, 318 (2014), 1-12
[7] Indra R., Moserova M., Sulc M., et al.: Neuro Endocrinology Letters, 34 Suppl. 2 (2013), 55-63
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MODIFICATION OF CARBON ELECTRODE WITH
GRAPHENE OXIDE SHEETS
Jana VLACHOVA1,2
, Lukas RICHTERA1,2
, David HYNEK1,2
and Rene KIZEK1,2*
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1,
613 00 Brno, Czech Republic
2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno,
Czech Republic
Abstract
In the current study, graphene oxide was used to enhance electrochemical determination of
adenine. The graphene oxide was mixed with Mg2+
ions to create Mg2+
-GO sheets. These
sheets were deposited on graphite electrode by electrophoretic deposition. The optimization of
electrophoretic deposition was carried out by changing of applied voltage in the range from 5
to 150 V and modified electrodes were compared using scanning electron microscopy. The
most thin and uniform layer of graphene oxide was achieved at small potential while too high
potential creates thick layers which inclined to peeling off. Electrochemical determination of
adenine was carried out using differential pulse voltammetry. Obtained results confirmed
findings from scanning electron microscopy and showed increase in signal about twice higher,
as well as an increase in sensitivity, for modified electrode than for unmodified one.
1. INTRODUCTION
Graphene oxide (GO) is a carbon-based material which creates a monolayer of carbon atoms
in form of hexagonal lattice with oxygenated functionalities [1]. Due oxygenated groups, the
GO is hydrophilic [2] and can be easily modified by various small molecules or polymers to
improve electrical, thermal and mechanical properties of graphene oxide [1]. These unique
properties of graphene oxide have been widely used for various applications especially in
electrochemistry for chemical sensor and biosensors [3,4].
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2. MATERIAL AND METHODS
The GO was prepared according to the Hummers method [5] following chemical exfoliation.
Mg2+
-GO sheets for electrophoretic deposition (EPD) was prepared by mixing of GO and
Mg(NO3)2 · 6H2O in weight ration 1:1. Isopropanol was used as a solvent. The final
concentration of GO in suspension was 0.05 mg/ml. The graphite and platinum electrode were
immersed into Mg2+
-GO sheets suspension. The distance between electrodes, applied voltage
and the deposition time was 10 mm, 10 V and 10 minutes, respectively. The negative charge
was applied on graphite electrode. The electrode modified by Mg2+
-GO sheets was observed
with scanning electron microscopy (SEM) (FE Tescan Mira II LMU) under the following
conditions: high vacuum mode (10–3 Pa), voltage of 15 kV. Electrochemical detection was
performed using Autolab IME 663 (Eco Chemie, Netherlands). Ag/AgCl/3M KCl was used as
a reference electrode, platinum as an auxiliary electrode and the graphite electrode modified
with GO-Mg2+
sheets was used as the working electrode. Electrochemical determination of
adenine was carried out by differential pulse voltammetry (DPV) in the presence of acetate
buffer (pH 5.0). DPV parameters: initial potential 0.2 V, end potential 1.4 V, potential step
0.005 V, modulation amplitude 0.025 V and modulation time 0.05 s.
3. RESULTS AND DISCUSSION
The optimization of EPD was carried out by application of voltage from 5 to 150 V. The
distance between electrodes and the deposition time was not changed. The best
electrophoretic deposition was achieved at small potential 5 and 10 V (Figure 1A) which
creates thin and uniform layer while too high potential 100 and 150 V creates thick layers
which inclined to peeling off (Figure 1B). DPV was performed with 3.3 µM adenine.
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Figure 1.: (A) graphite electrode modified with Mg2+
-GO sheets under 10 V (work distance 7 mm); (B) graphite
electrode modified with Mg2+
-GO sheets under 150 V (work distance 15 mm), (C) optimization of EPD of Mg2+
-
GO sheets under 5 - 150 V; (D) calibration curve of adenine on bare graphite electrode and graphite electrode
modified by Mg2+
-GO sheets.
Characteristic peak was situated at 0.9 V. The highest signals were obtained for 5 V and 10 V,
lower signal for 50 V and no signal was observed at 100 V and 150 V (Figure 1C). For the
comparison of detection abilities of bare graphite electrode and graphite electrode modified
with Mg2+
-GO sheets, applied voltage of 10 V was selected (Figure 1D). Calibration curves of
modified and unmodified electrode were fitted by linear regression and analytical parameters
such as regression parameters, standard deviation, limit of detection and limit of
quantification were calculated (Table 1). From obtained results it is evident that better
sensitivity of graphite electrode modified with Mg2+
-GO sheets was reached, on the other
hand analytical parameters show better detection limit of bare graphite electrode. This is a
consequence of dissimilar surface of modified electrode caused high standard deviation which
resulted in decrease of detection limit.
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Table 1: Analytical parameters of electrochemical detection of unmodified graphite electrode and
graphite electrode modified by Mg2+
-GO sheets, n = 3
Electrode Regression
equation
Linear
dynamic
range
(μM)
Linear
dynamic
range
(μg/ml)
R21
LOD2
(μM)
LOD
(µg/ml)
LOQ3
(μM)
LOQ
(µg/ml)
RSD4
(%)
Graphite y = 0.2163x - 0.1 1.48-
7.50
0.20-
1.01 0.9898 0.58 0.08 1.94 0.26 18.63
GO
sheets y = 0.4392x + 0.04
0.66-
7.50
0.09-
1.01 0.9949 0.83 0.11 2.75 0.37 39.47
1…regression coefficients, 2…limits of detection of detector (3 S/N), 3… limits of quantification of detector (10
S/N), 4…relative standard deviations
4. CONCLUSION
In present study, the graphite electrode was modified by Mg2+
-GO sheets using EPD. Due to
this modification we achieved increase of obtained signal and sensitivity for detection of
adenine.
5. ACKNOWLEDGEMENT
The work has been supported by NanoBioTECell P102/11/1068.
6. REFERENCES
[1] Dreyer D. R., Park S., Bielawski C. W. and Ruoff R. S.: Chemical Society Reviews, 39 (2010), 228.
[2] Zhu Y., Murali S., Cai W., Li X., Suk J. W., Potts J. R. and Ruoff R. S.: Advanced Materials, 22 (2010),
3906.
[3] Shao Y., Wang J., Wu H., Liu J., Aksay I. A. and Lin Y.: Electroanalysis, 22 (2010), 1027.
[4] Sharma P., Tuteja S. K., Bhalla V., Shekhawat G., Dravid V. P. and Suri C. R.: Biosensors and
Bioelectronics, 39 (2013), 99.
[5] Hummers W. S. and Offeman R. E.: Journal of the American Chemical Society, 80 (1958), 1339.
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GET TO KNOW METROHM
Peter BARATH*
Metrohm Czech Republic, s.r. o.; Na Harfě 935/5c; CZ –190 00 Prague 9; Czech Republic
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UV-VIS SPECTROPHOTOMETRY OF MICROLITER SAMPLE
VOLUME BY MEANS OF NANODROP INSTRUMENTS (THERMO
SCIENTIFIC)
Lucie KRAJCAROVÁ*
Pragolab s.r.o., Nad Krocínkou 55/285, 190 00 Praha 9; office Brno: Jamborova 32/3181, 615 00 Brno;
[email protected], www. pragolab.cz
Thermo Scientific NanoDrop family are smart, simple and robust instruments for UV-Vis
(NanoDrop 2000, NanoDrop 2000c, NanoDrop 8000, Fig. 1) or fluorescent (NanoDrop 3300)
measurements. They allow to analyze sample volumes as small as 0.5 μl, which is ideal for
precious high concentration samples. Higher concentration measurement capability up to
15,000 ng/μl (dsDNA) delivers accurate answers over a wide dynamic range without making
dilutions. NanoDrops measure a sample in less than 5 seconds. It is very easy to work with
NanoDrop - simply pipette a sample onto the pedestal and measure, then wipe the pedestal
and move to the next sample (Fig. 2a, 2b). Easy‑to‑clean polished stainless steel surfaces
assure that there is no cross contamination. The flexible software makes it easy to analyze
data and share results. Here we report on the capabilities of such instruments and demonstrate
examples of applications such as quantification of gold nanoparticles.
Figure 1: Thermo Scientific NanoDrop family.
Figure 2: All NanoDrop products utilize a unique technology that allows a sample to be pipetted directly onto an
optical measurement surface. The system uses inherent surface tension to hold a micro-volume sample in place
during the measurement cycle. Once the measurement is complete, the surfaces are simply wiped with a lint-free
lab wipe. a) Pipetting of 1 µl sample on pedestal b) microdrop measurement
Eco Chemie – Metrohm Autolab
Eco Chemie was founded in 1986 and is since 1999 a member of the Metrohm group of
companies. In 2009 the company name changed to Metrohm Autolab to reflect the customer
oriented combination of the world- wide Metrohm sales and support organization and the
high quality Autolab series of instruments developed by Eco Chemie. Metrohm Autolab is an
ISO9001 certified company. Metrohm Autolab based in Utrecht, The Netherlands, designs
and manufactures Autolab instruments, acces- sories, and software for electrochemistry.
Known for innovation, the Autolab was the first commer cial digital potentiostat/galvanostat
that was completely computer controlled. Our latest software package NOVA has again set a
high standard for powerful electrochemical research software. With our background and
knowledge in electrochemistry and our worldwide distributor network, our mission is to
serve the research community all over the world by supplying state of the art instruments
and unrivalled support. All Metrohm Autolab instruments are covered by a three year factory
warranty.
• Founded in 1986
• Based in Utrecht, The Netherlands
• Since 1999 part of the Metrohm Group
• Introduced the first computer controlled potentiostat/galvanostat
• Develops and produces the high quality Autolab range of products
• Strong background in electrochemistry
• Supported by the worldwide Metrohm distribution network
• Three years factory warranty on all instruments
• Dedicated to research
Book of abstracts
XV. Workshop of Physical Chemists and Electrochemists
Editor: Libuše Trnková
Technical adjustment: Iveta Pilařová
Published by Masaryk University, Brno 2015
1th
edition
ISBN 978-80-210-7857-4