· 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

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

[email protected]

(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

*[email protected]

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:

*[email protected].

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

*[email protected]

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

*[email protected]

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

*[email protected]

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.


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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.


25

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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

*[email protected]

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].


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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.


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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


29

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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

*[email protected]

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.


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


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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

*[email protected]

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


34

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2015

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

*[email protected]

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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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.


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).


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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.


38

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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

*[email protected]

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.


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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.


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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

*[email protected]

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.


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].


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).


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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


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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


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].


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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

*[email protected]

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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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.


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

*[email protected]

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.


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)


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

* [email protected]

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

*[email protected]

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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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.


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

*[email protected]

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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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].


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

*[email protected]

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.


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.


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


* [email protected]

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.


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.


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


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

*[email protected]

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].


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.


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

*[email protected]

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.


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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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


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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

*[email protected]

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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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.


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

*[email protected]

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.


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].


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,



Czech Republic

*[email protected]

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


*[email protected]

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].


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.


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

*[email protected]

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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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.


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


95

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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*




Czech Republic

*[email protected]

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.


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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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

* [email protected]

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.


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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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*


Republic

2 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health,

King’s College London, United Kingdom

*[email protected]

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.


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].


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


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

*[email protected]

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.


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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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,


*[email protected]

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.

mailto:[email protected]


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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.


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).


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*




Czech Republic

*[email protected]

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*


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

*[email protected]

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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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.


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




Czech Republic

* [email protected]

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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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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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

http://www.sciencedirect.com/science/journal/09565663

http://www.sciencedirect.com/science/journal/00219797


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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

*[email protected]

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.


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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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

* [email protected]

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


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*



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


Czech Republic

*[email protected]

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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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.


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

*[email protected]

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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*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

* [email protected]

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.


Quartz crystal microbalance devices were coated with continuous nanocrystalline diamond

layer (~300 nm thick) grown by pulsed linear antenna microwave plasma chemical vapour

mailto:*[email protected]





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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.


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


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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




Czech Republic

*[email protected]

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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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]


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

*[email protected]

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).


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.


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


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*




Czech Republic

*[email protected]

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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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.


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*




Czech Republic

*[email protected]

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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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.


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*


Czech Republic

* [email protected]

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.


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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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


*[email protected]

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.


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


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


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

*[email protected]

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.


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].


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


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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*




Czech Republic

*[email protected]

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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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.


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

*[email protected]


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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

*[email protected]

Thermo Scientific NanoDrop family are smart, simple and robust instruments for UV-Vis

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measurements. They allow to analyze sample volumes as small as 0.5 μl, which is ideal for

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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

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lab wipe. a) Pipetting of 1 µl sample on pedestal b) microdrop measurement

mailto:[email protected]


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 worldwide Metrohm sales and support organization and the

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Known for innovation, the Autolab was the first commer cial digital potentiostat/galvanostat

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Book of abstracts


Editor: Libuše Trnková

Technical adjustment: Iveta Pilařová

Published by Masaryk University, Brno 2015

1th

edition

ISBN 978-80-210-7857-4

· xv. workshop of physical chemists and electrochemists 4 brno 2015 the organization hosting the...

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